Preparation method and product of a thrombelastogram rapid detection cup reagent

Abstract

The application discloses a preparation method and product of a thromboelastography rapid detection cup reagent, and comprises the following steps: adding purified water into washed or calcined kaolin, adding glycine, mannitol, dextran and bovine serum albumin while stirring, and then adding a Tris-HCl system to adjust the pH to 6.5-7.8; finally, adding a recombinant lipidized tissue factor and uniformly stirring to obtain the reagent; wherein, the content of the washed or calcined kaolin is 0.01-0.8% based on 100% of the mass of each raw material, the content of the glycine is 2-6%, the content of the mannitol is 4-8%, the content of the dextran is 2-6%, the content of the bovine serum albumin is 0.2-5%, the content of the recombinant lipidized tissue factor is 0.2-1.5%, and the balance is made up to 100% by the purified water. The application provides a preparation method of the thromboelastography rapid detection cup reagent, and the prepared reagent can be stored for a long time, has good stability, and has good freeze-drying form and solubility.

Description

A method and product for preparing a rapid thromboelastography test cup reagent. Technical Field

[0001] This invention belongs to the field of in vitro diagnostic reagent technology, specifically relating to a method and product for preparing a rapid thromboelastography test cup reagent. Background Technology

[0002] Currently, rapid thromboelastography (TEG) testing cups are used in emergency departments, ICUs, anesthesiology departments, blood transfusion departments, obstetrics departments, stroke centers, and chest pain centers to assess patients' coagulation function, component blood transfusions, and the efficacy of anticoagulant drugs, offering significant advantages over conventional thromboelastography (TEG) cup testing. Conventional thromboelastography (TEG) cup testing currently takes approximately 35-60 minutes per sample, which is relatively long. In critically ill patients requiring emergency blood transfusions or during intraoperative assessments of coagulation function, conventional cups cannot quickly determine the overall coagulation function, thus limiting their effectiveness due to time constraints.

[0003] The thromboelastography rapid test cup utilizes the principle of simultaneously activating endogenous and exogenous pathways. In normal individuals, the activation time for coagulation factors using the rapid cup is 80-121 seconds, while that of the regular cup takes 5-10 minutes. Therefore, the rapid cup offers a shorter testing time, requiring only 3-20 minutes per sample, allowing for rapid assessment of a patient's overall dynamic coagulation function. Its test results exhibit superior stability, accuracy, and interference resistance compared to the regular cup. The results, presented in a graphical and data format, are easier to interpret. Parameters such as ACT, R, K, Angle, MA, and LY30 are available within minutes, promptly reflecting the dynamic functional status of coagulation factors and platelets. The rapid cup requires less blood; the minimum blood volume for a single rapid cup test is 340 μL, while the regular cup requires 1000 μL.

[0004] The principle behind the thromboelastography rapid detection cup technology is based on a new cellular coagulation theory. Whole blood testing not only reflects the interaction of coagulation components but also the participation of cells and some tissue factors in coagulation. The detection process simulates and records the entire dynamic process of intravascular coagulation initiation, clot formation, and fibrinolysis using a small amount of whole blood. It comprehensively assesses coagulation status by monitoring indicators such as coagulation factor activity, fibrinogen levels, platelet count, and fibrinolytic status. It can monitor both coagulation and fibrinolysis processes, and the ACT value sensitively reflects the anticoagulation status after heparin administration.

[0005] Compared to other testing methods, the advantages of the thromboelastography rapid test cup are that it can more quickly reflect the overall picture of coagulation, is simple to operate, and takes less time. It is especially suitable for emergency or resuscitation patients, and can quickly find the cause of bleeding in patients, prompting clinicians to provide effective blood transfusions and targeted resuscitation treatment for emergency patients. It can be widely used in many fields such as liver transplantation, heart transplantation, kidney transplantation, trauma surgery, and emergency surgery to guide blood transfusion, drug application, diagnosis, thrombolysis and anticoagulation therapy in emergency patients during surgery. Literature reports (Kashu JL, Moore EE, Wohlauer M, et al. Initial experiences with point-of-care rapid thrombelastography for management of life-threatening post injury coagulopathy[J]. Transfusion, 2012, 52(1): 23-33. DOI: 10.1111 / j.1537-2995.2011.03264; Xia Libo, Jian Cui, Zhang Biyu, et al. Establishment of reference intervals for thromboelastography in children and comparison with routine coagulation items[J]. Laboratory Medicine, 2017, 32(2): 81-85. Thromboelastography can better predict transfusion needs and solve the problems of when, what, and how much to transfuse in component transfusion therapy, providing a scientific, objective, and reliable basis for clinicians and blood bank personnel in selecting and guiding the use of blood products.

[0006] However, the reagents for thromboelastography testing cups in current technologies have problems such as short shelf life, poor stability, and poor solubility. Summary of the Invention

[0007] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0008] In view of the problems existing in the above and / or prior art, the present invention is proposed.

[0009] Therefore, the purpose of this invention is to overcome the shortcomings of the prior art and provide a method for preparing a rapid thromboelastography detection cup reagent.

[0010] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a method for preparing a rapid thromboelastography detection cup reagent, comprising,

[0011] Add purified water to washed or calcined kaolin, and while stirring, add glycine, mannitol, dextran and bovine serum albumin, then add Tris-HCl system to adjust the pH to 6.5-7.8;

[0012] Then add recombinant lipid-modifying tissue factor, stir well, and the reagent is obtained; wherein,

[0013] Based on the mass of each raw material as 100%, the content of the washed or calcined kaolin is 0.01-0.8%, the content of glycine is 2-6%, the content of mannitol is 4-8%, the content of dextran is 2-6%, the content of bovine serum albumin is 0.2-5%, the content of recombinant lipid-modified human tissue factor is 0.2-1.5%, and the balance is purified water to make up to 100%.

[0014] As a preferred embodiment of the preparation method described in this invention, the washed or calcined kaolin has a particle size of 0.01–0.25 μm.

[0015] As a preferred embodiment of the preparation method described in this invention, it further includes:

[0016] After stirring the reagent, draw the reagent into the bottom of a vial and stopper it for later use.

[0017] In a preferred embodiment of the preparation method described in this invention, the stirring speed is 200-400 r / min and the stirring time is 5-20 min.

[0018] As a preferred embodiment of the preparation method described in this invention, the method further includes filling and vacuum freeze-drying, specifically the following steps:

[0019] Pre-freezing: The pre-freezing temperature is -40 to -55℃, the cooling time is 1 to 3 hours, and the temperature is maintained for 2 to 6 hours after reaching the pre-freezing temperature;

[0020] Evacuation: Evacuate the chamber to a vacuum level of 50-200 ubar;

[0021] The reagent is dried.

[0022] As a preferred embodiment of the preparation method described in this invention, the drying includes:

[0023] After evacuation, raise the temperature to -20 to -30°C for 1 to 4 hours, and maintain the evacuation temperature at -20 to -30°C for 4 to 8 hours.

[0024] Raise the temperature to -10 to -20°C for 1 to 3 hours, and maintain the evacuation at -10 to -20°C for 2 to 6 hours.

[0025] Raise the temperature to 0-15℃ for 2-5 hours, and maintain the evacuation at 0-15℃ for 2-4 hours.

[0026] Raise the temperature to 20-30°C for 2-4 hours, reduce the vacuum to 5-30 ubar, and maintain the evacuation at 20-30°C for 3-6 hours to obtain the dried reagent.

[0027] Another objective of this invention is to overcome the shortcomings of the prior art and provide a product obtained by a method for preparing a rapid thromboelastography test cup reagent.

[0028] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a product obtained by a method for preparing a rapid thromboelastography test cup reagent, the product comprising washed or calcined kaolin, glycine, mannitol, dextran, bovine serum albumin, recombinant lipid-modified human tissue factor, and purified water; wherein,

[0029] Based on the mass of each raw material as 100%, the content of the washed or calcined kaolin is 0.01-0.8%, the content of glycine is 2-6%, the content of mannitol is 4-8%, the content of dextran is 2-6%, the content of bovine serum albumin is 0.2-5%, the content of recombinant lipid-modified human tissue factor is 0.2-1.5%, and the balance is purified water to make up to 100%.

[0030] Beneficial effects of this invention:

[0031] (1) The present invention provides a method for preparing a rapid thromboelastography test cup reagent. The prepared reagent can be stored for a long time and has good stability, lyophilized form and good solubility.

[0032] (2) The reagent formulation of the present invention contains non-animal tissue factors. Compared with the prior art, the main components are animal-derived tissue factors and phospholipids that require lipidation treatment. The non-animal-derived tissue factors of the present invention are easy to obtain, simple to prepare, and have good stability.

[0033] (3) The thromboelastography rapid detection cup reagent prepared by the present invention using vacuum freeze-drying technology has good freeze-dried form, good solubility and reconstitution stability, which is significantly better than the existing technology. Attached Figure Description

[0034] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein:

[0035] Figure 1 is a freeze-dried morphology diagram of the sample obtained in an embodiment of the present invention. Detailed Implementation

[0036] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0037] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0038] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0039] The recombinant lipid-modified human tissue factor used in this invention is a microbial metabolite, a commercially available product, purchased from Beijing Bosheng Technology Co., Ltd., product batch B211123, model BRP133.

[0040] The prior art reagent 1 in this invention was purchased from Beijing Lepu Medical Technology Co., Ltd., with product batch number 21SH06008 and production date of 2021 / 09 / 06;

[0041] The prior art reagent 2 in this invention was purchased from Chongqing Dingrun Medical Device Co., Ltd., with product batch number: 20211101 and production date: 20211112;

[0042] The prior art reagent 3 in this invention was purchased from Guangzhou Yangpu Medical Technology Co., Ltd., with product batch number 20210601 and production date 20210624.

[0043] Example 1

[0044] This embodiment provides a method for preparing a rapid thromboelastography test cup reagent, the main steps of which are:

[0045] (1) Add purified water to the washed or calcined kaolin, and while stirring, add glycine, mannitol, dextran and bovine serum albumin, and then add Tris-HCl system to adjust the pH to 6.5-7.8;

[0046] Then add recombinant lipid-modified human tissue factor, stir well, and the reagent is obtained; wherein,

[0047] Based on the mass of each raw material as 100%, the content of the washed or calcined kaolin is 0.2%, the content of glycine is 2%, the content of mannitol is 4%, the content of dextran is 2%, the content of bovine serum albumin is 0.3%, the content of recombinant lipid-modified human tissue factor is 0.2%, and the balance is purified water to make up to 100%.

[0048] (2) Filling of reagents for rapid detection of thallium force diagram:

[0049] After stirring the reagent obtained in step (1) at 250 r / min for 5 min, fill the vial and use a pipette to draw 20 μL of reagent into the bottom of the vial and stopper it for later use.

[0050] (3) Vacuum freeze-drying of reagent filling for rapid detection of thromboelastography cups:

[0051] Pre-freezing: The pre-freezing temperature is -40℃, the cooling time is 1 hour, and the temperature is maintained for 3 hours after reaching the pre-freezing temperature;

[0052] Evacuation: Evacuate the chamber to a vacuum level of 150 ubar;

[0053] Drying: Raise the temperature to -20℃ for 1 hour, and maintain the -20℃ temperature under vacuum for 5 hours;

[0054] Raise the temperature to -10℃ over 2 hours, and maintain the -10℃ temperature under vacuum for 4 hours.

[0055] Raise the temperature to 10℃ and the heating time is 2 hours. Maintain the temperature at 10℃ and evacuate for 2 hours.

[0056] The temperature was raised to 20°C for 2 hours, the vacuum level was reduced to 20 ubar, and the temperature was maintained at 20°C for 4 hours. The freeze-dried morphology of the resulting sample is shown in Figure 1.

[0057] This embodiment compares with existing technologies in terms of long-term storage stability at 2-8℃:

[0058] The reagents prepared in this embodiment and those in the prior art were placed at 2-8℃ and removed at 3, 9, 12, 18, and 24 months, respectively. Thromboelastography quality control level I was used on a JJ-8000 thromboelastography instrument (equipment serial number 20210501001), and the parameters ACT, R, K, Angle, and MA were recorded. The method is as follows: 1 ml of purified water was added to dissolve the quality control, 20 μL of 0.2 mol / L calcium chloride and 10 μL of the reagents of this invention or the prior art were added to the sample cup of the thromboelastography instrument, and then 340 μL of the quality control solution was added. After the detection, the average value and CV% of each parameter were calculated. The long-term stability of this invention and the prior art was investigated, as shown in Table 1.

[0059] Table 1

[0060]

[0061] The mean and coefficient of variation of various parameters of the present invention and the prior art after long-term storage at 2-8℃ for 24 months are shown in Table 2.

[0062] Table 2

[0063]

[0064] As shown in Table 1, the range of thromboelastography quality control level I is: ACT 80~118 s, R 0.2-0.8 min, K 0.8~1.1 min, Angle 74~82 deg, MA 64~72 mm.

[0065] The data above shows that, under 24 long-term stability tests at 2-8℃, the reagent of this invention exhibits superior long-term stability compared to existing reagents. The key parameter ACT (Active Chance) over 24 months indicates that while existing reagents are effective for up to 12 months, this invention can be effectively preserved for over 24 months. A lower CV% indicates less reagent variation and better stability; the CV% of all parameters in this invention is superior to that of existing technologies.

[0066] Example 2

[0067] Stability study after redissolution at room temperature:

[0068] The reagents of this invention were reconstituted with purified water and placed at room temperature. The thromboelastography quality control level I was used to detect the reagents at 2, 6, 9, and 12 hours after reconstitution, as shown in Table 3.

[0069] Table 3

[0070]

[0071] As shown in Table 3, the accuracy and stability of the present invention remain good after reconstitution and storage at room temperature for 12 hours, indicating that the present invention is effective for use within 12 hours at room temperature after reconstitution. In contrast, existing technologies 1, 2, and 3 have some parameters exceeding the quality control range after 6 hours of reconstitution, and their usability is only within 2 hours.

[0072] Example 3

[0073] Compared with Example 1, this embodiment has a different reagent formulation, but all other conditions are the same as in Example 1. The specific differences are as follows:

[0074] Based on the mass of each raw material as 100%, the content of the washed or calcined kaolin is 0.2%, the content of the glycine is 4%, the content of the dextran is 4%, the content of the bovine serum albumin is 0.3%, the content of the recombinant lipid-modified human tissue factor is 0.2%, and the balance is purified water to make up to 100%.

[0075] Example 4

[0076] Compared with Example 1, this embodiment has a different reagent formulation, but all other conditions are the same as in Example 1. The specific differences are as follows:

[0077] Based on the mass of each raw material as 100%, the content of the washed or calcined kaolin is 0.2%, the content of mannitol is 5%, the content of dextran is 3%, the content of bovine serum albumin is 0.3%, the content of recombinant lipid-modified human tissue factor is 0.2%, and the balance is purified water to make up to 100%.

[0078] Example 5

[0079] Compared with Example 1, this embodiment has a different reagent formulation, but all other conditions are the same as in Example 1. The specific differences are as follows:

[0080] Based on the mass of each raw material as 100%, the content of the washed or calcined kaolin is 0.2%, the content of glycine is 2%, the content of mannitol is 4%, the content of dextran is 2%, the content of bovine serum albumin is 0.5%, and the balance is purified water to make up to 100%.

[0081] The long-term stability of Examples 1, 3-5 was examined, and the results are shown in Table 4.

[0082] Table 4

[0083]

[0084] As shown in Table 4, Example 5, without the addition of recombinant lipo-modified human tissue factor, could not simultaneously activate intrinsic and extrinsic coagulation pathways, resulting in prolonged ACT and detection parameters falling outside the quality control range. Therefore, recombinant lipo-modified human tissue factor is a key raw material of this invention.

[0085] In Examples 3 and 4, no mannitol and glycine were added, and after 12 months of storage, the reagents gradually lost their activity. Example 1 illustrates that the combined use of mannitol and glycine in this invention provides good stabilizing and protective effects.

[0086] Comparative Example 1

[0087] Compared with Example 1, this embodiment has a different reagent formulation, but all other conditions are the same as in Example 1. Specifically:

[0088] Experiment 1: The content of recombinant lipid-modified human tissue factor was 0.4%;

[0089] Experiment 2: The content of recombinant lipid-modified human tissue factor was 0.8%;

[0090] Experiment 3: The content of recombinant lipid-modified human tissue factor was 1.2%;

[0091] Experiment 4: The content of recombinant lipid-modified human tissue factor was 1.4%.

[0092] The stability results are shown in Table 5.

[0093] Table 5

[0094]

[0095] In summary, this invention provides a method for preparing a rapid thromboelastography test cup reagent. The prepared reagent can be stored for a long time and has good stability, lyophilized form, and solubility.

[0096] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for preparing a rapid thromboelastography test cup reagent, characterized in that: The process involves adding purified water to washed or calcined kaolin, while stirring, adding glycine, mannitol, dextran, and bovine serum albumin, then adjusting the pH to 6.5–7.8 using a Tris-HCl system; finally, adding recombinant lipid-modified human tissue factor and stirring until homogeneous to obtain the reagent. The reagent comprises, by weight, 0.2% of the washed or calcined kaolin, 2% of the glycine, 4% of the mannitol, 2% of the dextran, 0.3% of the bovine serum albumin, and 0.2% of the recombinant lipid-modified human tissue factor, with the remainder being purified water to bring the total to 100%.

2. The preparation method according to claim 1, characterized in that: The washed or calcined kaolin has a particle size of 0.01–0.25 μm.

3. The preparation method according to claim 1 or 2, characterized in that: It also includes stirring the reagent, drawing the reagent into the bottom of a vial, and sealing it for later use.

4. The preparation method according to claim 3, characterized in that: The stirring is performed at a speed of 200–400 r / min for a duration of 5–20 min.

5. The preparation method according to any one of claims 1, 2, or 4, characterized in that: It also includes filling and vacuum freeze drying, with the following steps: pre-freezing: the pre-freezing temperature is -40 to -55℃, the cooling time is 1 to 3 hours, and the temperature is maintained for 2 to 6 hours after reaching the pre-freezing temperature; evacuation: the vacuum degree in the chamber is evacuated to 50 to 200 ubar; drying to obtain the dried reagent.

6. The preparation method according to claim 5, characterized in that: The drying process includes: after evacuation, raising the temperature to -20 to -30°C for 1 to 4 hours, maintaining the temperature at -20 to -30°C under evacuation for 4 to 8 hours; raising the temperature to -10 to -20°C for 1 to 3 hours, maintaining the temperature at -10 to -20°C under evacuation for 2 to 6 hours; raising the temperature to 0 to 15°C for 2 to 5 hours, maintaining the temperature at 0 to 15°C under evacuation for 2 to 4 hours; raising the temperature to 20 to 30°C for 2 to 4 hours, reducing the vacuum to 5 to 30 ubar, maintaining the temperature at 20 to 30°C under evacuation for 3 to 6 hours, thereby obtaining the dried reagent.

7. The product obtained by any one of the preparation methods according to claims 1 to 6.

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