Thromboelastography coagulation activation test reagent and application thereof

Abstract

This invention relates to the field of in vitro coagulation detection technology, specifically to a thromboelastography coagulation activation assay reagent and its application. The coagulation activation assay reagent provided by this invention comprises a buffer solution, an intrinsic coagulation activator, tissue factor, a stabilizer, and a preservative; the stabilizer includes three types: component 1, component 2, and component 3. This invention effectively improves the stability of the assay reagent by using a combination of three types of stabilizers, avoiding the problem of failure during long-term storage and transportation. Due to the improved reagent stability, kaolin and tissue factor can effectively activate both intrinsic and intrinsic coagulation activation pathways, ensuring the accuracy of thromboelastography detection.

Description

Technical Field

[0001] This invention relates to the field of in vitro coagulation detection technology, specifically to a thromboelastography coagulation activation detection reagent and its application. Background Technology

[0002] Normal coagulation function is essential for human life. Assessing blood flow and coagulation is a crucial indicator for clinical treatment and disease prevention. Traditional coagulation tests provide a static reflection of blood clot formation, only showing a portion of the coagulation cascade and failing to fully reflect the patient's true coagulation status. This is a significant cause of treatment delays and inappropriate procedures. Thromboelastography (TEG) is a novel cytology-based coagulation testing modality. Using whole blood in vitro, it converts changes in viscoelasticity during blood coagulation into digital signals using physical methods, thus recording them as a coagulation curve that reflects the complete picture of coagulation.

[0003] Thromboelastography (TEG) has become the most important indicator for monitoring coagulation function during the perioperative period. Conventional TEG primarily activates the intrinsic coagulation pathway, and the testing process is time-consuming, making it less suitable for patients requiring emergency surgery. Rapid TEG (r-TEG) ​​adds tissue factor to the conventional TEG, simultaneously activating both intrinsic and extrinsic coagulation pathways, shortening the coagulation activation time, and rapidly completing the coagulation test of whole blood in vitro. Rapid TEG has significant application value in assessing the overall coagulation profile, surgical treatment of trauma patients, early diagnosis of hyperfibrinolysis, prediction of massive transfusion and thrombosis risks, and guiding massive transfusions. Rapid TEG is widely used in trauma surgery, liver transplantation, and cardiac surgery, and has crucial application value in monitoring perioperative coagulation function, guiding clinical transfusion strategies, and scientifically managing blood products.

[0004] Currently, there are few coagulation activation test kits available on the domestic and international markets. These kits are expensive, have high testing costs, short stability times, and require use with custom-made instruments, while also exhibiting high specificity. Therefore, developing a rapid thromboelastography kit with high stability is an urgent task for us. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a thromboelastography coagulation activation detection reagent and its application. The coagulation activation detection reagent provided by this invention has high stability, effectively avoiding the problem of failure during long-term storage and transportation.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] The coagulation activation assay reagent provided by this invention includes a buffer solution, an intrinsic coagulation activator, a tissue factor, a stabilizer, and a preservative.

[0008] The stabilizer comprises component 1, component 2 and component 3;

[0009] Component 1 is one or a mixture of several of silica gel, agar, chitosan, cellulose, carboxymethyl cellulose, and alginate;

[0010] Component 2 is glutaraldehyde;

[0011] Component 3 is one or a mixture of several of ethylene glycol, glycerol, tea polyphenols, sorbitol and apple polyphenols;

[0012] The endogenous activator includes one or more of kaolin, ellagic acid, and diatomaceous earth.

[0013] The coagulation activation test reagent provided by this invention has good uniformity, excellent stability, and is not prone to precipitation. The coagulation activation test reagent is used in conjunction with a thromboelastography instrument to detect relevant indicators.

[0014] In some embodiments, the buffer solution includes Tris buffer, HEPES buffer, and PBS buffer; in some specific embodiments, the buffer solution is Tris buffer. In this invention, the buffer solution is prepared from water and a buffering component. The buffering component includes at least one of Tris, HEPES, and PBS.

[0015] In some embodiments, the preservative includes one or more of sodium azide, thimerosal, Kathon, gentamicin sulfate, and Proclin 300. In some specific embodiments, the preservative is gentamicin sulfate. The gentamicin sulfate used in this invention is an aminoglycoside drug with strong antibacterial activity against *Pseudomonas aeruginosa*, *Proteus* spp., *Escherichia coli*, *Klebsiella* spp., *Enterobacter* spp., *Serratia* spp., *Shigella* spp., *Citrobacter* spp., *Neisseria*, and *Staphylococcus aureus*.

[0016] In some embodiments, the coagulation activation assay reagent of the present invention includes Tris, kaolin, tissue factor, stabilizer, preservative, and water. The stabilizer includes a mixture of carboxymethyl cellulose, glutaraldehyde, and apple polyphenols. Specifically, the present invention first utilizes the bifunctional reagent glutaraldehyde to form covalent bonds between proteins of tissue factor. The principle is that glutaraldehyde has two aldehyde groups, both of which can react with the free amino groups of proteins or enzymes to form Schiff bases. Second, by changing conditions (temperature, pH), carboxymethyl cellulose is made into a gel-like substance, which can be achieved through hydrophobic interactions, hydrogen bonding, etc. Third, the gel-like cellulose has a good encapsulation capacity for active substances, further diffusing the tissue factor complex into the carrier through diffusion. In addition, apple polyphenols are selected as one of the stabilizer components. Apple polyphenols are a type of plant polyphenol. The active phenolic hydroxyl groups in plant polyphenol molecules have antioxidant properties and can release hydrogen ions, thereby disrupting the terminating oxidation chain reaction. Similarly, apple polyphenols have a much greater scavenging capacity for singlet oxygen, hydrogen peroxide, and hydroxyl radicals than vitamin C. It allows the reagent to maintain its original activity even at higher temperatures. Experiments show that the combination of the three stabilizers can significantly improve the stability of the detection reagent.

[0017] Furthermore, this invention optimized and screened the components and concentrations of the coagulation activation assay reagent. The results showed that when the concentrations of Tris, kaolin, tissue factor, stabilizer, and preservative were 0.1%~0.6% (w / v) Tris, 0.001%~0.01% (w / v) kaolin, 0.1%~1% (v / v) tissue factor, 0.01%~0.2% (w / v) carboxymethyl cellulose, 0.01%~0.5% (v / v) glutaraldehyde, 0.5%~2.0% (v / v) apple polyphenols, and 0.002~0.01% (w / v) preservative, the stability and repeatability were high, and the detection effect was good. In some preferred embodiments, the coagulation activation assay reagent comprises water and components at the following concentrations: 0.36% (w / v) Tris, 0.005% (w / v) kaolin, 0.25% (v / v) tissue factor, 0.01%~0.25% (v / v) glutaraldehyde, 0.01%~0.05% (w / v) carboxymethyl cellulose, 0.5%~2% (w / v) apple polyphenols, and 0.005% (w / v) preservative. The optimal detection results are achieved when the concentrations of each component are 0.36% (w / v) Tris, 0.005% (w / v) kaolin, 0.25% (v / v) tissue factor, 0.05% (w / v) carboxymethyl cellulose, 0.25% (v / v) glutaraldehyde, 0.9% (v / v) apple polyphenols, and 0.005% (w / v) preservative.

[0018] The coagulation activation assay reagent provided by this invention has a pH value of 7.0 to 8.0. Specifically, in some embodiments, the pH value of the coagulation activation assay reagent is 7.0, 7.4, or 8.0.

[0019] Specifically, in the specific embodiments of this invention, the exogenous coagulation activator used is recombinant human tissue factor esterified product. This invention uses recombinant human tissue factor esterified product, combined with carboxymethyl cellulose, glutaraldehyde, and apple polyphenols, which greatly enhances the stability of tissue factor.

[0020] In a specific embodiment of the present invention, the kaolin is nano-sized powdered kaolin, and the preservative is gentamicin sulfate.

[0021] This invention uses nano-sized powdered kaolin and human recombinant tissue factor lipids, which can simultaneously activate the human body's endogenous and extrinsic coagulation pathways, accelerate the coagulation process, and enable the blood to coagulate rapidly.

[0022] In some specific embodiments, the coagulation activation test reagent provided by the present invention is composed of water and components of the following concentrations: 0.005% kaolin, 0.25% recombinant human tissue factor ester, 0.01% glutaraldehyde, 0.01% carboxymethyl cellulose, 0.5% apple polyphenols, 0.36% Tris, 0.005% gentamicin sulfate and the balance being water, pH=7.4;

[0023] It may be composed of water and 0.005% kaolin, 0.25% recombinant human tissue factor ester, 0.25% glutaraldehyde, 0.05% carboxymethyl cellulose, 2% apple polyphenols, 0.36% Tris, 0.005% gentamicin sulfate and the balance water, pH=7.4;

[0024] It may be composed of water and 0.005% kaolin, 0.25% recombinant human tissue factor ester, 0.25% glutaraldehyde, 0.05% carboxymethyl cellulose, 0.9% apple polyphenols, 0.36% Tris, 0.005% gentamicin sulfate and the balance water, pH=7.4.

[0025] The present invention also provides the application of the coagulation activation test reagent in the preparation of a coagulation activation test kit.

[0026] The present invention also provides a coagulation activation test kit, which includes the coagulation activation test reagent described in the present invention and calcium chloride.

[0027] Furthermore, the concentration of calcium chloride can be 0.2~0.8 mol / L, specifically 0.2 mol / L, 0.3 mol / L, 0.4 mol / L, 0.5 mol / L, 0.6 mol / L, 0.7 mol / L, or 0.8 mol / L.

[0028] In the coagulation activation assay reagent provided by this invention, the percentage content "%" of Tris, kaolin, carboxymethyl cellulose, apple polyphenols, and preservatives are all expressed as % (w / v), where w / v is the mass-volume ratio in g / mL. The percentage concentration "%" of tissue factor and bifunctional cross-linking agent is expressed as a volume percentage.

[0029] The coagulation activation assay reagent provided by this invention comprises a buffer solution, an endogenous coagulation activator, tissue factor, a stabilizer, and a preservative. The stabilizer comprises components 1, 2, and 3. Component 1 is a mixture of one or more of silica gel, agar, chitosan, cellulose, carboxymethyl cellulose, and alginate. Component 2 is selected as glutaraldehyde. Component 3 is a mixture of one or more of ethylene glycol, glycerol, tea polyphenols, sorbitol, and apple polyphenols. The endogenous activator comprises one or more of kaolin, ellagic acid, and diatomaceous earth. This invention, by employing the combination of the above three stabilizers, effectively improves the stability and repeatability of the assay reagent, effectively avoiding the problem of coagulation activation assay reagent failure during long-term storage and transportation. Detailed Implementation

[0030] This invention provides a thromboelastography coagulation activation detection reagent and its application. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0031] The test materials used in this invention are all common commercial products and can be purchased on the market.

[0032] The present invention will be further illustrated below with reference to the embodiments:

[0033] Example 1

[0034] The coagulation activation assay reagent consists of 0.005% kaolin, 0.25% recombinant human tissue factor ester, 0.01% glutaraldehyde, 0.01% carboxymethyl cellulose, 0.5% apple polyphenols, 0.36% Tris, 0.005% gentamicin sulfate, and the balance being water (pH=7.4).

[0035] Example 2

[0036] The coagulation activation assay reagent consists of 0.005% kaolin, 0.25% recombinant human tissue factor ester, 0.25% glutaraldehyde, 0.05% carboxymethyl cellulose, 2% apple polyphenols, 0.36% Tris, 0.005% gentamicin sulfate, and the balance being water (pH=7.4).

[0037] Example 3

[0038] The coagulation activation assay reagent consists of 0.005% kaolin, 0.25% recombinant human tissue factor ester, 0.25% glutaraldehyde, 0.05% carboxymethyl cellulose, 0.9% apple polyphenols, 0.36% Tris, 0.005% gentamicin sulfate, and the balance being water (pH=7.4).

[0039] Comparative Example 1

[0040] The stabilizer consists of glutaraldehyde and carboxymethyl cellulose. The components of the coagulation activation assay reagent are as follows by percentage: 0.01% kaolin, 5% recombinant human tissue factor ester, 0.5% glutaraldehyde, 0.02% carboxymethyl cellulose, 0.36% Tris, 0.005% gentamicin sulfate, and the balance being water (pH=7.0).

[0041] Comparative Example 2

[0042] The stabilizer is composed of glutaraldehyde and carboxymethyl cellulose. The components of the coagulation activation assay reagent are as follows by percentage: 0.001% kaolin, 0.1% recombinant human tissue factor ester, 0.5% glutaraldehyde, 0.02% carboxymethyl cellulose, 0.36% Tris, 0.005% gentamicin sulfate and the balance water (pH=7.0).

[0043] Comparative Example 3

[0044] The stabilizer is composed of glutaraldehyde and carboxymethyl cellulose. The components of the coagulation activation assay reagent are as follows (in percentage): 0.005% kaolin, 0.25% recombinant human tissue factor ester, 0.5% glutaraldehyde, 0.02% carboxymethyl cellulose, 0.36% Tris, 0.005% gentamicin sulfate, and the balance being water (pH=7.0).

[0045] The amounts of components other than water in the detection reagents of Examples 1-3 and Comparative Examples 1-7 are shown in Table 1:

[0046] Table 1

[0047]

[0048] Comparative Example 4

[0049] The coagulation activation assay reagent, by percentage, consists of: 0.005% kaolin, 0.25% recombinant human tissue factor ester, 0.36% Tris, 0.005% gentamicin sulfate, and the balance being water. It contains no stabilizers.

[0050] Comparative Example 5

[0051] One existing technology is a thromboelastography coagulation activation test kit (coagulation method), specifically the thromboelastography coagulation activation test kit (coagulation method) from Shenzhen Maiketian Biomedical Technology Co., Ltd.

[0052] Comparative Example 6

[0053] The stabilizer consists of glutaraldehyde and carboxymethyl cellulose, and the coagulation activation assay reagent consists of the following percentages: 0.005% kaolin, 0.25% recombinant human tissue factor ester, 0.25% glutaraldehyde, 0.05% carboxymethyl cellulose, 0.36% Tris, 0.005% gentamicin sulfate, and the balance being water (pH=7.4).

[0054] Comparative Example 7

[0055] The stabilizer is apple polyphenols, and the coagulation activation assay reagent consists of the following percentages: 0.005% kaolin, 0.25% recombinant human tissue factor ester, 0.9% apple polyphenols, 0.36% Tris, 0.005% gentamicin sulfate, and the balance being water (pH=7.4).

[0056] In the comparative examples above, the concentrations of glutaraldehyde and recombinant human tissue factor esters are expressed as volume percentages (%). The concentrations of other components are expressed as weight-volume (w / v) percentages, with the specific unit being g / mL.

[0057] Test Example 1 Feasibility Analysis

[0058] Blood samples from the same source were tested using the detection reagents of Examples 1-3 and Comparative Examples 1-7. The detection process included the following steps:

[0059] 1. On the corresponding thromboelastography software interface, enter the patient's name, test name, and select "CRT-Citrated Rapid TEG" from the drop-down box for the test type.

[0060] 2. Mount a set of sample cups on each channel of the thromboelastography instrument.

[0061] 3. Transfer 20 μL of calcium chloride reagent to the bottom of the sample cup.

[0062] 4. Pipette the indicated volume of reconstituted solvent into the coagulation activation test reagent bottle to reconstitute the lyophilized powder reagent.

[0063] 5. Gently shake the activator vial until the activator is completely reconstituted, then add 10 μL of coagulation activation reagent to the bottom of the reaction vessel.

[0064] 6. Transfer 340 μL of blood into the sample cup.

[0065] 7. Move the cup to the test position and begin the test.

[0066] 8. The coagulation process takes about 20 minutes to complete; if fibrinolysis is to be observed, it may take up to 1 hour.

[0067] 9. After the test is completed, click "Stop" or "Stop" on the software interface, remove the disposable cup, and dispose of it according to laboratory requirements.

[0068] The test results are shown in Table 2:

[0069] ACT value: Activation time for coagulation, a parameter specific to rapid thromboelastography. ACT is generally considered to represent the time from the start of testing to the initial fibrin formation.

[0070] K value: This parameter is generally considered to represent the amplification stage of thrombin activation, which mainly leads to the solidification of blood clots through fibrinogen cleavage and fibrin polymerization. K time and Angle angle are generated simultaneously. In clinical practice, Angle often replaces K value because Angle value is more meaningful than K value.

[0071] Angle value: Reflects the combined effect of fibrinogen and platelets when a blood clot begins to form, at which point fibrinogen is the dominant function, and is an indicator for detecting fibrinogen function.

[0072] MA value: reflects the maximum strength of the forming blood clot and the stability of the blood clot formation. It is mainly affected by two factors: platelets and fibrin. Among them, the role of platelets is greater than that of fibrin, accounting for about 80%.

[0073] Table 2

[0074]

[0075] Results: The detection results of the test reagent of the present invention (Examples 1-3) are similar to those of commercially available test reagents (Comparative Example 5), indicating that the test reagent of the present invention can be effectively applied to thromboelastography.

[0076] Furthermore, the stability and repeatability of the detection reagent of the present invention were investigated.

[0077] Test Example 2 Stability Assessment

[0078] The stability assessment process is as follows:

[0079] 1. The detection reagents in Examples 3-6 and Comparative Examples 3-7 were placed at 37°C for accelerated stabilization for 4 weeks.

[0080] 2. Take reagents on days 0, 7, 14, and 28 after accelerated stabilization at 37℃ and test the quality control of the same batch.

[0081] The same batch of quality control samples were tested using the test reagents from Examples 1-3 and Comparative Examples 3-7, respectively. The test results are shown in Table 3.

[0082] Relative deviation (%) = [(day n - day 0) / day 0] × 100%

[0083] Table 3

[0084]

[0085] Table 4

[0086]

[0087] Results: Example 3, using a stabilizer combination of carboxymethyl cellulose, glutaraldehyde, and apple polyphenols, remained stable for four weeks at 37°C, with minimal deviation in the detection of the same batch of samples. Examples 1-2 showed reduced activation capacity and increased ACT compared to Example 3, with other parameters exhibiting varying degrees of change, resulting in slightly worse performance than Example 3. Comparative Example 4, lacking stabilizer components, rapidly deactivated within four weeks due to the absence of any substances affecting the stability of kaolin and tissue factors. Comparative Example 5 showed minimal changes in detection results, but its stability was worse than Example 3. Comparative Examples 3 and 6, lacking apple polyphenols, exhibited worse stability than Comparative Example 5; Comparative Example 7, lacking glutaraldehyde and carboxymethyl cellulose, showed even worse stability, though slightly better than Comparative Example 1. In summary, the stability of Examples 1-3 of this invention was significantly higher than that of Comparative Examples 4-7, with Example 3 showing the best detection performance.

[0088] Test Example 3 Repeatability Evaluation

[0089] The repeatability assessment process is as follows:

[0090] The test reagents from Example 3 and Comparative Example 5 were used to perform ten quality control tests on the same batch over a period of 28 days.

[0091] The same batch of quality control samples were tested using the test reagents from Example 3 and Comparative Example 5, respectively. The test results are shown in Table 5.

[0092] Table 5

[0093]

[0094] Results: As shown in the table, Example 3 was accelerated at 37°C for 4 weeks, and the CV of the same batch of samples was very small in 10 tests. The CV of Example 3 was smaller than that of Comparative Example 5, indicating that Example 3 has better repeatability and uniformity and is superior to commercially available test reagents.

[0095] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications 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 coagulation activation test reagent, characterized in that, Including Tris buffer, kaolin, tissue factor, stabilizer and preservative; The stabilizer is composed of carboxymethyl cellulose, glutaraldehyde, and apple polyphenols; The concentrations of each component in the coagulation activation assay reagent are as follows: 0.1%~0.6% (w / v) Tris buffer, 0.001%~0.01% (w / v) kaolin, 0.1%~1% (v / v) tissue factor, 0.01%~0.05% (w / v) carboxymethyl cellulose, 0.01%~0.25% (w / v) glutaraldehyde, 0.5%~2.0% (w / v) apple polyphenols and 0.002%~0.01% (w / v) preservative.

2. The coagulation activation test reagent according to claim 1, characterized in that, The preservatives include one or more of sodium azide, thimerosal, Kathon, gentamicin, and Proclin 300.

3. The coagulation activation test reagent according to claim 1, characterized in that, The coagulation activation assay reagent comprises water and components of the following concentrations: 0.36% (w / v) Tris buffer, 0.005% (w / v) kaolin, 0.25% (v / v) tissue factor, 0.01%~0.25% (v / v) glutaraldehyde, 0.01%~0.05% (w / v) carboxymethyl cellulose, 0.5%~2% (w / v) apple polyphenols and 0.005% (w / v) preservative.

4. The coagulation activation test reagent according to claim 1, characterized in that, Its pH is 7.0~8.

0.

5. The coagulation activation test reagent according to any one of claims 1 to 4, characterized in that, The coagulation activation assay reagent consists of the following components at the following concentrations: 0.005% kaolin, 0.25% recombinant human tissue factor ester, 0.01% glutaraldehyde, 0.01% carboxymethyl cellulose, 0.5% apple polyphenols, 0.36% Tris, 0.005% gentamicin sulfate, and the balance being water, pH=7.4; It may be composed of 0.005% kaolin, 0.25% human recombinant tissue factor ester, 0.25% glutaraldehyde, 0.05% carboxymethyl cellulose, 2% apple polyphenols, 0.36% Tris, 0.005% gentamicin sulfate and the balance water, pH=7.4; It may be composed of 0.005% kaolin, 0.25% recombinant human tissue factor ester, 0.25% glutaraldehyde, 0.05% carboxymethyl cellulose, 0.9% apple polyphenols, 0.36% Tris, 0.005% gentamicin sulfate and the balance water, pH=7.

4.

6. The use of the coagulation activation test reagent according to any one of claims 1 to 5 in the preparation of a coagulation activation test kit.

7. A coagulation activation test kit, characterized in that, It includes the coagulation activation test reagent and calcium chloride as described in any one of claims 1 to 5.

8. The coagulation activation test kit according to claim 7, characterized in that, The concentration of calcium chloride is 0.2~0.8 mol / L.

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