Thromboelastography quality control product and preparation method, application in preparation of coagulation detection product, coagulation detection product and coagulation detection method

Abstract

The application belongs to the technical field of in-vitro diagnostic reagents, and particularly relates to thrombelastography quality control products and a preparation method and application in preparation of coagulation detection products, a coagulation detection product and a coagulation detection method. The application realizes high adhesion of quality control product coagulation parameters and clinical actual measurement values by optimizing plasma proportioning and screening special excipients, can simulate six typical clinical coagulation states from normal coagulation to low coagulation, high coagulation, fibrinogen abnormality, DIC and the like, and simultaneously has the characteristics of simple preparation process and excellent long-term stability.

Description

Technical Field

[0001] This invention belongs to the field of in vitro diagnostic reagent technology, specifically relating to thromboelastography quality control products and preparation methods, their application in the preparation of coagulation testing products, coagulation testing products, and coagulation testing methods. Background Technology

[0002] Thromboelastography (TEG) is a technique for dynamically monitoring the entire coagulation process and is widely used in clinical settings such as surgery, trauma, and liver transplantation for coagulation function assessment. The accuracy of its test results directly depends on the stability of the instrument, reagents, and operating system; therefore, reliable quality control materials are required for routine calibration and quality control.

[0003] Most thromboelastography quality control products on the market are currently bilevel (e.g., normal / abnormal) or trilevel (normal / hypercoagulable / hypocoagulable), and most use a single animal plasma (e.g., porcine plasma) as the matrix. These quality control products have the following problems:

[0004] Incomplete level coverage: It is difficult to simultaneously simulate multiple coagulation states that may occur in clinical practice (such as hypercoagulability, hypocoagulability, fibrinogen abnormalities, etc.).

[0005] Single matrix: The composition of single animal plasma differs from that of human plasma, and the coagulation factor profile has a low degree of matching with that of humans, which may lead to insufficient stability of quality control products;

[0006] Low parameter differentiation: The R value and MA value of some quality control products overlap, making it impossible to effectively distinguish different coagulation states;

[0007] The preparation process is complex: some methods require the separate extraction of clotting factors or platelets, which involves complicated steps.

[0008] To address the aforementioned issues, this invention develops a multi-level, multi-matrix, highly discriminative, easy-to-prepare, and highly stable thromboelastography quality control product. Through multi-source animal plasma compounding and screening of special excipients, it achieves accurate simulation of clinical coagulation status, solving the technical pain points of existing products. Summary of the Invention

[0009] In view of this, the technical problem to be solved by the present invention is to provide a thromboelastography quality control product.

[0010] This invention provides a thromboelastography quality control product, comprising a plasma matrix and a lyophilization protectant;

[0011] This may include plasma matrix, anticoagulants, and lyophilization protectants;

[0012] This may include plasma matrix, fibrinogen matrix, and lyophilization protectant;

[0013] It may include plasma matrix, fibrinogen matrix, lyophilization protectant and anticoagulant.

[0014] In some embodiments, the plasma matrix is ​​composed of porcine plasma, bovine plasma, sheep plasma, and rabbit plasma. Experimental verification showed that, compared to quality control products prepared from single-plasma, two-plasma, or three-plasma combinations, the quality control product prepared from four-plasma combinations exhibited better repeatability, with CV values ​​for all parameters within 10%.

[0015] In some embodiments, the fibrinogen matrix comprises fibrinogen and amino acid compounds. Compared to the various macromolecular protective agents, colloids, and potent anticoagulants required in traditional formulations, amino acid compounds greatly simplify the formulation while achieving functional upgrades, protecting proteins and fine-tuning coagulation dynamics.

[0016] In some embodiments, the lyophilization protectant includes at least one of 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), a polyhydroxy compound, a polymer, a saccharide compound, taurine, or serum albumin. This lyophilization protectant provides a more stable lyophilization system during the preparation of thromboelastography quality control samples, maximizing the stability, sensitivity, and specificity of the lyophilized thromboelastography quality control samples, thereby achieving more accurate technical results.

[0017] In some embodiments, the anticoagulant includes sodium heparin, sodium enoxaparin, nadroparin, or sodium dalteparin.

[0018] In some embodiments, the amino acid compound in the fibrinogen matrix includes glycine and / or arginine;

[0019] In the freeze-drying protectant, the polyhydroxy compound includes mannitol, the polymer includes polyethylene glycol (PEG) or polyvinylpyrrolidone (PVP), the saccharide compound includes trehalose or sucrose, and the serum albumin includes bovine serum albumin or human serum albumin.

[0020] Compared to other reagents, the above components work closely together and are highly compatible, enabling them to be used more effectively in the preparation of thromboelastography quality control products, thereby achieving more accurate technical results.

[0021] In some specific embodiments, the amino acid compounds in the fibrinogen matrix are glycine and arginine;

[0022] In the freeze-drying protectant, the polyhydroxy compound is mannitol, the polymer is polyethylene glycol (PEG), the saccharide compound is trehalose, and the serum albumin is human serum albumin.

[0023] The above-mentioned components, through experimental screening and optimized compatibility, form a synergistic composite system. Compared with the single or simple combination of protectants and stabilizers in traditional formulations, this invention achieves functional upgrades while significantly simplifying the formulation through the synergistic effect of multiple components. The quality control products prepared using the component system described in this invention show a reduction of more than 35% in the attenuation rate of key coagulation parameters compared with traditional formulations, and the inter-batch coefficient of variation (CV) is stably controlled within 3%, demonstrating excellent repeatability and long-term stability. Among them, the specific combination of glycine and arginine has both protein protection and coagulation dynamics regulation functions, simplifying the formulation while enabling fine-tuning of key parameters such as R value and K value; trehalose forms hydrogen bonds with proteins through hydroxyl groups to replace water molecules, maintain the native conformation of proteins, and increase the glass transition temperature of the mixture, forming a stable amorphous glassy state, effectively preventing the collapse of freeze-dried products and protein denaturation; taurine, as an endogenous antioxidant, establishes a continuous reducing atmosphere in the solid phase environment, protecting protein sulfhydryl groups and sensitive amino acid residues from oxidative damage, ensuring that the coagulation parameters of the quality control product are highly stable within the shelf life; compared with the use of bovine serum albumin, human serum albumin has high homology with human clotting proteins, which can reduce the adsorption and denaturation of active proteins during the freeze-drying process, while eliminating heterologous immune interference and significantly improving the clinical consistency of pathological state simulation; polyethylene glycol (PEG) forms a spatial barrier through long-chain molecules to prevent protein aggregation and increases the glass transition temperature of the freeze-dried concentrate, so that the product can remain stable at higher temperatures, which is beneficial to freeze-drying process operation and finished product storage.

[0024] In some embodiments, the mass ratio of the plasma matrix, fibrinogen matrix, lyophilization protectant, and anticoagulant is (90~120):(0~0.5):(3~7):(0~0.1). For example, the mass ratio of the plasma matrix, fibrinogen matrix, lyophilization protectant, and anticoagulant is (100:0:5:0.005), (100:0.02:5:0.007), (100:0.1:5:0), (100:0:5:0), (100:0:5:0.025), (95:0.3:4:0.05), (115:0.4:6:0.08), or (120:0.02:5:0.1).

[0025] As a feasible example, the thromboelastography quality control product mentioned above,

[0026] The mass ratio of the plasma matrix, fibrinogen matrix, lyophilization protectant, and anticoagulant is 100:0:5:0.005;

[0027] The mass ratio of the plasma matrix, fibrinogen matrix, lyophilization protectant, and anticoagulant is 100:0.02:5:0.007;

[0028] The mass ratio of the plasma matrix, fibrinogen matrix, lyophilization protectant, and anticoagulant is 100:0.1:5:0;

[0029] The mass ratio of the plasma matrix, fibrinogen matrix, lyophilization protectant, and anticoagulant is 100:0:5:0;

[0030] The mass ratio of the plasma matrix, fibrinogen matrix, lyophilization protectant, and anticoagulant is 100:0:5:0.025.

[0031] In some embodiments, the mass ratio of porcine plasma, bovine plasma, sheep plasma, and rabbit plasma is (45~65):(5~25):(10~40):(10~30). For example, the mass ratio of porcine plasma, bovine plasma, sheep plasma, and rabbit plasma is (55:15:25:20), (50:5:30:15), (60:15:20:15), (55:25:10:10), (45:10:25:30), (65:15:15:10), (45:5:40:20), (50:20:30:15), or (60:15:10:20). Experimental optimization has determined that this mass ratio range has the best mixing effect compared to other mass ratio ranges. By adjusting the plasma ratio, precise control can be achieved over the four key parameters of thromboelastography—R value, K value, Angle angle, and MA value—thereby simulating six typical clinical coagulation states, including normal coagulation, hypocoagulation, hypercoagulation, fibrinogen abnormality, and DIC.

[0032] As a feasibility example, in the plasma matrix,

[0033] The mass ratio of the pig plasma, bovine plasma, sheep plasma and rabbit plasma is 55:15:25:20. The coagulation parameters of the quality control product under this ratio are within the range of normal human thromboelastography test results, and each parameter is close to the median value.

[0034] The mass ratio of pig plasma, bovine plasma, sheep plasma and rabbit plasma was 50:5:30:15. The quality control sample under this ratio showed a hypocoagulable state, with extremely low MA value and R, K and Angle values ​​basically in the normal or near-normal range, indicating platelet function defect or hypofibrinogenemia.

[0035] The mass ratio of the pig plasma, bovine plasma, sheep plasma and rabbit plasma is 60:15:20:15. Under this ratio, the coagulation parameters of the quality control product are all near the upper and lower limits of normal values, and the MA value covers the lower limit of normal and most normal values. It can simulate the normal or borderline normal state, and can also reflect mild thrombocytopenia or mild functional impairment.

[0036] The mass ratio of the pig plasma, bovine plasma, sheep plasma and rabbit plasma is 55:25:10:10. The quality control product under this ratio exhibits a hypercoagulable state, with an extremely short R value, an increased Angle angle and an increased MA value, which shows a highly dynamic state of extremely fast coagulation initiation and rapid and firm blood clot formation.

[0037] The mass ratio of the porcine plasma, bovine plasma, sheep plasma, and rabbit plasma is 45:10:25:30. The quality control product under this ratio exhibits paradoxical coagulation characteristics, with a significantly shortened R value and a significantly reduced MA value. This can simulate the consumptive hypocoagulable phase of disseminated intravascular coagulation (DIC) or reflect a hypercoagulable state combined with thrombocytopenia and hyperfibrinolysis.

[0038] The mass ratio of the pig plasma, bovine plasma, sheep plasma, and rabbit plasma is 65:15:15:10; the quality control sample under this ratio exhibits a mild hypercoagulable or coagulation activated state, with the R value at the lower limit of normal and the MA value being low or slightly reduced within the normal range.

[0039] Alternatively, the mass ratio of the pig plasma, bovine plasma, sheep plasma, and rabbit plasma is 45:5:40:20. The quality control sample under this ratio exhibits significant hypocoagulability characteristics, with a significantly prolonged R value, prolonged K value, and decreased Angle angle, while the MA value is normal or only slightly reduced, indicating a serious impairment in the coagulation initiation process, but the strength of the blood clot is still acceptable after formation.

[0040] In some embodiments, the mass ratio of fibrinogen, glycine, and arginine is (0.1~1.5):(0.5~2):(0.1~1); for example, the mass ratio of fibrinogen, glycine, and arginine is 0.5:1:0.5, 0.4:0.5:0.1, 0.45:0.8:0.3, 0.6:1.5:0.8, 0.8:2:1, or 1.5:1.8:1.

[0041] In some embodiments, the mass ratio of fibrinogen, glycine, and arginine is 0.5:(0.5~2):(0.1~1); for example, the mass ratio of fibrinogen, glycine, and arginine is 0.5:1:0.5, 0.5:0.5:0.1, 0.5:0.8:0.3, 0.5:1.5:0.8, or 0.5:2:1. This range was determined through optimized screening. By adjusting the mass ratio of glycine to arginine, the Angle value and R value can be independently and linearly controlled over a wide range. For example, with a fixed arginine concentration, the Angle value decreases linearly with increasing glycine concentration; with a fixed glycine concentration, the R value increases linearly with increasing arginine concentration.

[0042] In some specific embodiments, the mass ratio of fibrinogen, glycine, and arginine is 0.5:1:0.5.

[0043] In some embodiments, the mass ratio of 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), mannitol, polyethylene glycol (PEG), trehalose, taurine, and human serum albumin is (0.01~0.15):(1~7):(3~7):(3~7):(2~6):(1~3); for example, the mass ratio of 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), mannitol, polyethylene glycol (PEG), trehalose, taurine, and human serum albumin is (0.1:2:3:5:3:1.5), (0.1:2:5:5:3:1.5), (0.1:4:5:5:4:2), (0.1:6:5:5:5:2.5), (0.15:3:5:5:4.5:2.3), or (0.08:5:6:6:5:2.5). This mass ratio range was determined through optimization and screening. Compared with other ratio ranges, the quality control products prepared using this range have batch-to-batch coefficients of variation (CV) of less than 10% for R value, K value, Angle angle, and MA value, demonstrating excellent stability and repeatability.

[0044] As a feasibility example, the mass ratio of 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), mannitol, polyethylene glycol (PEG), trehalose, taurine, and human serum albumin is 0.1:2:3:5:3:1.5;

[0045] The mass ratio of 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), mannitol, polyethylene glycol (PEG), trehalose, taurine, and human serum albumin is 0.1:2:5:5:3:1.5.

[0046] The mass ratio of 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), mannitol, polyethylene glycol (PEG), trehalose, taurine, and human serum albumin is 0.1:4:5:5:4:2.

[0047] Alternatively, the mass ratio of the 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), mannitol, polyethylene glycol (PEG), trehalose, taurine, and human serum albumin may be 0.1:6:5:5:5:2.5.

[0048] Preferably, the mass ratio of 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), mannitol, polyethylene glycol (PEG), trehalose, taurine and human serum albumin is 0.1:4:5:5:4:2 or 0.1:6:5:5:5:2.5.

[0049] More preferably, the mass ratio of 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), mannitol, polyethylene glycol (PEG), trehalose, taurine, and human serum albumin is 0.1:4:5:5:4:2. Through extensive screening experiments, specific combinations and ratio ranges were determined, resulting in freeze-dried quality control products with CV values ​​of less than 10% for R, K, Angle angle, and MA, and exhibiting good stability in aging tests, while effectively saving costs. The effectiveness of this technology depends on the specific combination and the ratio range between the components; the absence of any one component or changes in the ratio will prevent the achievement of optimal results.

[0050] In some embodiments, the anticoagulant is dalteparin sodium. Compared to other anticoagulants (such as heparin sodium, enoxaparin sodium, or nadroparin), dalteparin sodium has a more balanced anti-Xa / anti-IIa ratio of approximately 2.0:1 to 2.5:1, resulting in a relatively stronger inhibitory effect on thrombin (factor IIa). This characteristic is of significant value in quality control products: when it is necessary to simulate a hypocoagulable state primarily caused by thrombin activity inhibition (such as patient samples treated with direct thrombin inhibitors), dalteparin sodium more closely approximates the coagulation characteristics of real samples, thereby improving the clinical relevance and coverage of the quality control product.

[0051] In some embodiments, the mass-volume fraction of the dalteparin sodium is 0.5% to 1.5%. For example, the mass-volume fraction of the dalteparin sodium is 0.5%, 0.8%, 1.0%, 1.3%, or 1.5%.

[0052] In some specific embodiments, the mass-volume fraction of the dalteparin sodium is 1%.

[0053] In some specific embodiments, the solvent in the 1% (w / v) dalteparin sodium solution is water.

[0054] In some embodiments, the thromboelastography quality control material, by weight, comprises:

[0055] The mixture contained 50 parts of pig plasma, 5 parts of bovine plasma, 30 parts of sheep plasma, 15 parts of rabbit plasma, 0.005 parts of dalteparin sodium, and 5 parts of lyophilization protectant. The lyophilization protectant contained 4-hydroxyethylpiperazine ethanesulfonic acid, mannitol, polyethylene glycol, trehalose, taurine, and human serum albumin in a mass ratio of 0.1:4:5:5:4:2.

[0056] The mixture contains 60 parts of porcine plasma, 15 parts of bovine plasma, 20 parts of sheep plasma, 15 parts of rabbit plasma, 0.02 parts of fibrinogen matrix, 0.007 parts of dalteparin sodium, and 5 parts of lyophilization protectant. The fibrinogen matrix contains fibrinogen, glycine, and arginine in a mass ratio of 0.5:1:0.5. The lyophilization protectant contains 4-hydroxyethylpiperazine ethanesulfonic acid, mannitol, polyethylene glycol, trehalose, taurine, and human serum albumin in a mass ratio of 0.1:4:5:5:4:2.

[0057] The mixture contained 55 parts of porcine plasma, 25 parts of bovine plasma, 10 parts of sheep plasma, 10 parts of rabbit plasma, 0.1 parts of fibrinogen matrix, and 5 parts of lyophilization protectant. The fibrinogen matrix contained fibrinogen, glycine, and arginine in a mass ratio of 0.5:1:0.5. The lyophilization protectant contained 4-hydroxyethylpiperazine ethanesulfonic acid, mannitol, polyethylene glycol, trehalose, taurine, and human serum albumin in a mass ratio of 0.1:4:5:5:4:2.

[0058] 45 parts of pig plasma, 10 parts of bovine plasma, 25 parts of sheep plasma, 30 parts of rabbit plasma, and 5 parts of lyophilization protectant, wherein the mass ratio of 4-hydroxyethylpiperazine ethanesulfonic acid, mannitol, polyethylene glycol, trehalose, taurine, and human serum albumin in the lyophilization protectant is 0.1:4:5:5:4:2.

[0059] The sample contained 65 parts of pig plasma, 15 parts of bovine plasma, 15 parts of sheep plasma, 10 parts of rabbit plasma, and 5 parts of lyophilization protectant. The lyophilization protectant contained 4-hydroxyethylpiperazine ethanesulfonic acid, mannitol, polyethylene glycol, trehalose, taurine, and human serum albumin in a mass ratio of 0.1:4:5:5:4:2.

[0060] Alternatively, 45 parts of pig plasma, 5 parts of bovine plasma, 40 parts of sheep plasma, 20 parts of rabbit plasma, 0.025 parts of dalteparin sodium, and 5 parts of lyophilization protectant, wherein the mass ratio of 4-hydroxyethylpiperazine ethanesulfonic acid, mannitol, polyethylene glycol, trehalose, taurine, and human serum albumin in the lyophilization protectant is 0.1:4:5:5:4:2.

[0061] In some embodiments, the quality control materials include quality control level A, quality control level B, quality control level C, quality control level D, quality control level E, and quality control level F;

[0062] The range of the quality control level A is R value 4~9min, K value 1~4min, Angle value 55~75°, MA value 25~40mm;

[0063] The range of the quality control level B is R value 4~10min, K value 1~4min, Angle value 55~75°, MA value 40~65mm;

[0064] The quality control level C ranges as follows: R value 1.5~2.5 min, K value 0.8~1.0 min, Angle value 72~92°, MA value 55~80 mm.

[0065] The quality control level D is defined as follows: R value 1.0~2.5 min, K value 0.5~2.2 min, Angle value 60~90°, MA value 20~40 mm.

[0066] The quality control level E is defined as follows: R value 2.5~5.0 min, K value 0.5~2.2 min, Angle value 60~90°, MA value 40~60 mm.

[0067] The quality control level F is defined as follows: R value > 10 min, K value > 3 min, Angle value < 55°, and MA value < 50 mm.

[0068] In some embodiments, the quality control product further includes preservatives and / or coagulation activators.

[0069] In some embodiments, the preservative includes potassium sorbate, gentamicin sulfate, Proclin 300, or sodium azide; the coagulation activator includes kaolin, tissue factor, or fibrinogen.

[0070] The present invention also provides a method for preparing the thromboelastography quality control product as described above, comprising: mixing plasma matrix, fibrinogen matrix, lyophilization protectant and anticoagulant, and then pre-freezing and sublimating at elevated temperature to obtain the product.

[0071] In some embodiments, the pre-freezing conditions include: the pre-freezing temperature is -45°C and the pre-freezing time is 6 hours.

[0072] In some embodiments, the conditions for the heating and sublimation include:

[0073] Under a vacuum of 150 ubar, the temperature was sequentially maintained at -40℃ for 4 h, -35℃ for 4 h, -30℃ for 3 h, -20℃ for 3 h, and -15℃ for 3 h.

[0074] Under a vacuum of 100 ubar, the temperature was maintained at -5℃ for 3 h and then at 0℃ for 3 h.

[0075] Under a vacuum of 80 ubar, the temperature was maintained at 5℃ for 3 h and then at 15℃ for 3 h, respectively.

[0076] Under a vacuum of 50 ubar, the temperature was maintained at 20°C for 3 h and then at 25°C for 4 h.

[0077] The pre-freezing conditions and sublimation heating conditions were obtained through experimental screening. Compared with other parameters, the R value, K value, Angle angle and MA value of the freeze-dried quality control products obtained using these parameters were basically consistent with the test results before freeze-drying, with small changes, good repeatability, and relative deviations of less than 0.1.

[0078] Furthermore, the present invention provides the application of the thromboelastography quality control product as described above, or the thromboelastography quality control product obtained by the preparation method described above, in the preparation of coagulation test products.

[0079] Furthermore, the present invention also provides coagulation testing products, including the thromboelastography quality control product as described above, or the thromboelastography quality control product obtained by the preparation method described above.

[0080] In some embodiments, the coagulation testing product includes a thromboelastography coagulation test reagent or kit.

[0081] Furthermore, the present invention provides a coagulation detection method, comprising: testing a sample to be tested using the coagulation detection product as described above.

[0082] This method can be used for both diagnostic and non-diagnostic purposes. Diagnostic applications include assessing a patient's coagulation function or assisting in the diagnosis of coagulation-related diseases (such as disseminated intravascular coagulation, hemophilia, coagulation disorders caused by liver disease, or monitoring the efficacy of anticoagulants). Non-diagnostic applications include research on coagulation mechanisms, drug screening, performance verification of quality control products, and the development and testing of new coagulation reagents or instruments.

[0083] The core innovation of this invention lies in:

[0084] 1) Optimization of Four-Source Composite Plasma Matrix: The composite matrix is ​​made by compounding the plasma of four animals, namely pig, cow, sheep and rabbit, in a specific optimized ratio (pig plasma 45%~65%, cow plasma 5%~25%, sheep plasma 10%~40%, rabbit plasma 10%~30%). It is prepared by adding modulating components such as anticoagulants, fibrinogen and heparin sodium, which effectively simulates the coagulation factor spectrum of human plasma and synergistically improves the stability and parameter adjustability of the quality control product.

[0085] 2) Highly Consistent with Clinical Practice: The key parameters of the quality control product, especially the R value range (4~10 min), are highly consistent with the actual test results (R value 5~10 min) of normal human blood samples tested using activated coagulation reagent in clinical practice, solving the technical problem of existing quality control product parameters being out of sync with clinical practice. The quality control product of this invention has a simple preparation process, excellent stability, and high clinical consistency, enabling precise and comprehensive quality control of a complete detection system including a thromboelastography instrument, activated coagulation reagent, and calcium chloride solution.

[0086] 3) Innovative combination of excipients: Special freeze-drying protectant, anticoagulant and fibrinogen solution were screened to achieve high activity retention after freeze-drying and reconstitution of quality control products. The CV value of key parameters was <10% after 28 days of accelerated aging at 37℃, and the long-term stability was excellent.

[0087] 4) Independently adjustable parameters: By adjusting the plasma ratio and the amount of excipients added, the four parameters R, K, Angle, and MA can be independently controlled to achieve accurate simulation of six coagulation states. Moreover, the preparation process is simple and does not require separate extraction of coagulation factors / platelets.

[0088] This invention relates to thromboelastography quality control products and their preparation methods, as well as their application in the preparation of coagulation testing products, coagulation testing products, and coagulation testing methods. By optimizing plasma ratios and screening specialized excipients (such as lyophilization protectants, anticoagulants, and fibrinogen solutions), this invention ensures that the coagulation parameters of the quality control products closely match clinically measured values, simulating six typical clinical coagulation states: normal coagulation, hypocoagulation, hypercoagulation, fibrinogen abnormalities, and disseminated intravascular coagulation (DIC). Furthermore, this quality control product features a simple preparation process and excellent long-term stability. Attached Figure Description

[0089] Figure 1 This is a schematic diagram of the output of thromboelastography. Detailed Implementation

[0090] This invention provides thromboelastography quality control materials and their preparation method, as well as their application in the preparation of coagulation testing products, coagulation testing products, and coagulation testing methods. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It is particularly important to note 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 clearly modify or appropriately change and combine 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.

[0091] Unless otherwise defined in this invention, the scientific and technical terms associated with this invention shall have the meanings understood by one of ordinary skill in the art.

[0092] The terms “comprising,” “including,” and “having” are used interchangeably to indicate the inclusiveness of a scheme, meaning that the scheme may contain elements other than those listed. It should also be understood that the use of “comprising,” “including,” and “having” herein also provides for schemes “consisting of…”.

[0093] When used herein, the term “and / or” includes the meaning of “and,” “or,” and “all or any other combination of elements linked by the term.”

[0094] The term "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single or multiple items.

[0095] It should be understood that in the various embodiments of this application, the order of the above processes does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0096] The numerical ranges and parameters involved in this invention have been presented as precisely as possible in the specific embodiments. However, any numerical value inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all numerical ranges or specific data used in this disclosure may have a reasonable deviation within a certain range, such as ±10%, ±5%, ±1%, or ±0.5%.

[0097] The core objective of this invention is to provide a thromboelastography level 6 quality control product based on a composite plasma matrix from pigs, cattle, sheep, and rabbits. By optimizing the plasma ratio and screening special excipients (lyophilization protectant, anticoagulant, and fibrinogen solution), the coagulation parameters of the quality control product closely match the clinical measured values. It can simulate six typical clinical coagulation states, from normal coagulation to hypocoagulation, hypercoagulation, fibrinogen abnormality, and DIC. At the same time, it has the characteristics of simple preparation process and excellent long-term stability.

[0098] Anticoagulant: Sodium citrate is the main anticoagulant used when collecting whole blood from animals.

[0099] The anticoagulant used during the commissioning process is selected from one of the following: heparin sodium (Sinopharm), enoxaparin sodium (Dalian Meilun), nadextrin (Ningbo Taisto Biotechnology), or dalteparin sodium (Maclean).

[0100] The freeze-drying protectant is selected from one or more of the following: trehalose (Aladdin), sucrose, arginine, glycine, mannitol, bovine serum albumin (BSA, Roche), PEG, PVP, taurine, or human serum albumin (HSA).

[0101] Preservatives include potassium sorbate, gentamicin sulfate, Proclin 300, or sodium azide (McCathy).

[0102] Coagulation activators include kaolin, tissue factor, or fibrinogen (Shenyang Baiying Biotechnology), etc.

[0103] Fibrinogen is derived from bovine plasma.

[0104] Selection Instructions:

[0105] (1) Dalteparin Sodium - Innovation Point 1

[0106] Enoxaparin sodium: The anti-Xa / anti-IIa ratio is approximately 3.3:1 to 5.3:1, with anti-Xa activity being dominant.

[0107] Dalteparin sodium: The anti-Xa / anti-IIa ratio is approximately 2.0:1 to 2.5:1, which is a more balanced ratio. This means that it has a relatively stronger inhibitory effect on thrombin (factor IIa).

[0108] Nadroparin has the highest anti-Xa / anti-IIa ratio, usually greater than 3.0:1, but this varies between different brands, and its anticoagulant activity may be more selective.

[0109] Advantages: This difference in anticoagulant activity profile is crucial in quality control products. For example, to simulate a hypocoagulable state primarily caused by inhibited thrombin activity (such as in patients treated with certain direct thrombin inhibitors), using dalteparin sodium, which has relatively stronger anti-IIa activity, may more closely approximate the real-world sample. This increases the clinical relevance and coverage of the quality control product.

[0110] (2) Specific amino acid combinations (such as glycine + arginine, which can both protect proteins and fine-tune coagulation dynamics) -- Innovation Point 2

[0111] (Glycine 0.5%~2%, Arginine 0.1%~1%)

[0112] The use of a specific amino acid combination of glycine and arginine in the preparation of thromboelastography quality control products is a highly innovative and functionally integrated design. It surpasses the single protective function of traditional lyophilization protectants, achieving the dual goals of "physical protection" and "biochemical fine-tuning".

[0113] Integrated solution: By using two clearly defined small-molecule amino acids, multiple large-molecule protective agents, colloids, and strong anticoagulants that may be required in traditional formulations are replaced, greatly simplifying and upgrading the formulation.

[0114] Independent adjustable parameters: Experiments have shown that by adjusting the molar ratio of glycine to arginine, the Angle and R values ​​can be independently and linearly controlled over a wide range (for example, with a fixed arginine concentration, the Angle value decreases linearly with increasing glycine concentration; with a fixed glycine concentration, the R value increases linearly with increasing arginine concentration), which is something that cannot be achieved with existing technologies.

[0115] Excellent stability: Quality control products protected by this combination show a reduction of more than 35% in the decay rate of key parameters and a CV value of less than 3% in long-term accelerated stability tests.

[0116] (3) Trehalose 5%

[0117] Core function: Preferred sugar protectant.

[0118] Mechanism: Through the "water displacement" theory, during protein dehydration, hydroxyl groups form hydrogen bonds with polar protein groups, replacing water molecules and maintaining the protein's native conformation. Simultaneously, it increases the glass transition temperature of the mixture, forming a stable amorphous glassy state and preventing lyophilized products from collapsing and protein denaturation.

[0119] Advantages: Excellent protective effect, chemically stable, non-reducing, and not prone to Maillard reaction.

[0120] (4) Taurine 4% - Innovation Point 3

[0121] The problem is that coagulation factors (especially II, V, VII, VIII, protein C, etc.), fibrinogen, and phospholipids are prone to oxidative modification during storage.

[0122] Mechanism of action of taurine:

[0123] Taurine is one of the strongest endogenous antioxidants in the body, and it can effectively scavenge hydroxyl radicals, hypochlorous acid, and other free radicals.

[0124] In the solid-phase environment of freeze-drying and storage, a continuous "reduced atmosphere" is established to protect protein thiol groups (-SH) and sensitive amino acid residues.

[0125] Effect: Ensures that the kinetics and endpoint parameters (R, K, Angle, MA) of the coagulation reaction are highly stable within the shelf life (e.g., 18-24 months) of the quality control product, with minimal batch-to-batch CV values.

[0126] (5) Human serum albumin 2% -- Innovation point 4 (Patent authorized by other manufacturers, more often BSA bovine serum albumin is used)

[0127] Core Functions: Protein stabilizer and "sacrificial agent." Excellent Functional Fidelity: The homology of HSA with human clotting proteins makes its protective behavior more closely resemble physiological states. Quality control products protected by HSA, when simulating pathological plasma conditions (such as heparinization and hypofibrinogenemia), show significantly improved consistency with real clinical samples in terms of abnormal parameters (such as prolonged R value and decreased MA value), while controls using BSA show deviations. Elimination of Foreign Interference Risk: Fundamentally eliminates the potential immune interference introduced by foreign proteins, ensuring the reliability of results when using quality control products to monitor samples from patients with various immune diseases, thus broadening the product's clinical applicability.

[0128] mechanism:

[0129] Surface activity: Reduces protein adsorption and denaturation at the gas-liquid interface and on container surfaces.

[0130] Preferred denaturation: Under freeze-drying stress, HSA, as an abundant and relatively stable protein, can preferentially withstand some denaturation stress, thereby "protecting" more precious, low-concentration active clotting proteins (such as fibrinogen and clotting factors).

[0131] Buffer and carrier: Provides a certain colloidal osmotic pressure and buffering capacity.

[0132] (6) Polyethylene glycol 5%

[0133] Core functions: polymer protectant and glass forming agent.

[0134] mechanism:

[0135] Spatially stable: Its long-chain molecules can form a physical barrier around the protein, preventing the protein from agglomerating during the concentration process.

[0136] Increasing Tg': Effectively increases the glass transition temperature of the freeze-dried concentrate, enabling the product to remain stable at higher temperatures, which is beneficial for freeze-drying process operation and finished product storage.

[0137] Impact: The type of PEG (such as PEG 4000, 8000) affects the effect. The larger the molecular weight, the stronger the glass transition ability, but excessively high viscosity may affect dispensing and reconstitution.

[0138] The test materials used in this invention are all common commercial products and can be purchased on the market.

[0139] Enoxaparin sodium was purchased from Dalian Meilun, nadextrin from Ningbo Taisto Biotechnology, dalteparin sodium from Maclean's, trehalose from Aladdin, bovine serum albumin (BSA) from Roche, sodium azide from McCarthy, fibrinogen from Shenyang Baiying Biotechnology, and the remaining reagents were purchased from Sinopharm Group.

[0140] The present invention will be further illustrated below with reference to the embodiments:

[0141] (I) Animal plasma screening and characterization

[0142] Thromboelastography and five coagulation parameters (fibrinogen, PT, APTT, FVII activity, and antithrombin activity) were used to test the coagulation characteristics, advantages, and disadvantages of each plasma. Based on literature review, the coagulation characteristics of each plasma were analyzed to provide a theoretical basis for compounding. The results are shown in Table 1.

[0143] Table 1. Analysis of plasma coagulation characteristics and advantages / disadvantages in four animal species.

[0144]

[0145] Where R is the coagulation reaction time, K is the clot formation time, Angle is the clot formation rate, and MA is the maximum clot strength.

[0146] Experimental results show that there are significant differences in coagulation properties of plasma from different species:

[0147] Pig plasma has a fibrinogen content close to that of human plasma, and is rich in coagulation factors with sufficient sources; however, the platelet membrane receptor structure differs from that of humans, antithrombin III activity is higher, and the coagulation balance deviates to some extent from that of humans.

[0148] Bovine plasma has high activity of coagulation factors VII and X, and good thermal stability of fibrinogen; however, it has a short prothrombin time (PT), which easily leads to a hypercoagulable background.

[0149] Sheep plasma has higher levels of coagulation factors IX and XI and moderate antithrombin activity; however, its fibrinogen content (2.8 g / L) is low, and its overall coagulation rate is slower than that of human plasma.

[0150] Rabbit plasma platelet phospholipid membrane composition is highly similar to that of humans, and it is highly sensitive to coagulation activators such as tissue factor; however, the total amount of coagulation factors is low, fibrinolysis is prone to occur, and antithrombin activity is low.

[0151] In summary, the coagulation properties of the four animal plasmas each have their own advantages and disadvantages, and no single plasma can completely mimic the coagulation characteristics of human plasma. Porcine plasma is closest to human plasma in terms of fibrinogen levels and polymerization rate; bovine plasma has high coagulation factor activity and good fibrinogen thermal stability; sheep plasma has high similarity to human coagulation kinetics; and rabbit plasma's platelet-related coagulation characteristics are more closely similar to human plasma. Through reasonable blending, it is hoped that precise regulation of coagulation performance can be achieved.

[0152] (II) Freeze-drying program screening experiment

[0153] Take 100% porcine blood plasma, dispense 1 ml / bottle into vials, and freeze-dry according to the three freeze-drying procedures in Table 2:

[0154] Table 2 Freeze-drying program settings

[0155]

[0156] After freeze-drying, the parameters of the quality control samples were tested using thromboelastography. The results are shown in Table 3. For programs 1 and 2, the R, K, Angle, and MA parameters of the quality control samples after freeze-drying changed significantly, with poor repeatability and relative deviations generally above 0.1. For program 3, the test results of the quality control samples after freeze-drying were similar to those of the porcine plasma before freeze-drying, with good repeatability and relative deviations all below 0.1.

[0157] Table 3. Test results after freeze-drying under different procedures

[0158]

[0159] (III) Component Screening Experiment

[0160] 1. Experimental design for quality control samples of multiple plasma compound preparations

[0161] Based on the thromboelastography and coagulation test results of the four types of plasma in Table 1, experiments were designed as shown in Table 4 to verify the stability of quality control products prepared by combining one type of plasma, two types of plasma, three types of plasma, and four types of plasma (using porcine plasma as a base, due to its high fibrinogen content and stable source) before and after freeze-drying.

[0162] Table 4. Experimental Design for Quality Control Samples of Multiple Plasma Compounds

[0163]

[0164] According to the above formula, lyophilization was carried out according to the same lyophilization procedure 3: pre-freezing temperature -45℃ for 6 hours; after pre-freezing, sublimation was carried out in a temperature gradient of 5~10℃, with vacuum controlled at 150 ubar, and held at -40℃, -35℃, -30℃, -20℃, and -15℃ for 4 hours, 4 hours, 3 hours, 3 hours, and 3 hours respectively; vacuum controlled at 100 ubar, and held at -5℃ and 0℃ for 3 hours and 3 hours respectively; vacuum controlled at 80 ubar, and held at 5℃ and 15℃ for 3 hours and 3 hours respectively; vacuum controlled at 50 ubar, and held at 20℃ and 25℃ for 3 hours and 4 hours respectively (hereinafter referred to as lyophilization procedure 3, and will not be described in detail again). After lyophilization, the repeatability was tested on a thromboelastography machine, and the results are shown in Table 5.

[0165] Table 5. Repeatability test results of quality control materials for multiple plasma compound preparations

[0166]

[0167] The test results show that after preparing quality control samples using single-plasma and two-plasma combinations, the CVs of R, K, Angle, and MA all exceeded 10% for one or two parameters. After preparing quality control samples using three-plasma combinations, the repeatability improved significantly, with some parameters exceeding 9% but less than 10%. After preparing quality control samples using four-plasma combinations, the CVs were all within 5%, showing better repeatability.

[0168] 2. Single-factor plasma regulatory effect test after four types of plasma were combined.

[0169] Using the median values ​​of thromboelastography parameters in normal individuals as the target (Target R=7.5min, Target K=2.0min, Target Angle=62.5°, Target MA=60.0mm), extensive experiments were conducted to determine that when 55% porcine plasma, 15% bovine plasma, 25% sheep plasma, and 20% rabbit plasma were used, the parameters R=7.4min, K=1.8min, Angle=63.7°, and MA=62mm were met. These values ​​are within the range of normal human thromboelastography test results and are very close to the median values.

[0170] Using pigs (55%), cattle (15%), sheep (25%), and rabbits (20%) as baselines, a single-factor variable experiment was conducted to verify the regulatory effects of each plasma component on TEG parameters. The core regulatory patterns are shown in Table 6.

[0171] Table 6 Single-factor variable experiment

[0172]

[0173] According to the above formula, lyophilization was carried out using the same lyophilization procedure 3. After lyophilization, thromboelastography was performed, and the results are shown in Table 7.

[0174] Table 7. Results of thromboelastography after freeze-drying

[0175]

[0176] Analysis of the test results shows that:

[0177] Increased bovine plasma levels lead to elevated MA and shortened R (procoagulant effect), which is a core regulatory component of hypercoagulable states.

[0178] Increasing sheep plasma levels leads to decreased MA value and prolonged R value (anticoagulant effect), which is a core regulatory component of hypocoagulable state;

[0179] Increasing rabbit plasma levels significantly shortens the R value (high sensitivity to coagulation activation), making it a core regulatory component for coagulation initiation rate;

[0180] Increasing porcine plasma provides a stable fibrinogen background, enhances the MA value base, and is a fundamental component of the complex matrix.

[0181] 3. Optimization experiment of compound plasma ratio

[0182] Based on the regulatory patterns of each plasma component on TEG parameters and extensive testing, the formulations of six quality control products were initially adjusted as shown in Table 8:

[0183] Table 8 Optimization of Compound Plasma Formulation

[0184]

[0185]

[0186] Based on the above formula, the test results before freeze-drying are shown in Table 9:

[0187] Table 9. Results of thromboelastography test before freeze-drying.

[0188]

[0189] The freeze-drying process was carried out using the same freeze-drying procedure 3. The test data before and after freeze-drying are shown in Table 10.

[0190] Table 10 Results of Thromboelastography Test after Freeze-Drying

[0191]

[0192] Lyophilization affected the coagulation parameters of six levels of plasma mixtures to varying degrees. Levels D and E showed good lyophilization tolerance, with minimal changes in coagulation parameters before and after lyophilization, remaining largely stable. Levels A, B, C, and F, however, exhibited varying degrees of parameter deviation, requiring formulation optimization through the addition of fibrinogen solution and anticoagulants: Levels A and F showed significantly shortened R values ​​(coagulation reaction time) after lyophilization, indicating enhanced coagulation activation tendency. It is proposed to add anticoagulants to the compound system to restore its expected hypocoagulable characteristics. Level B showed a decrease in both R and MA values ​​(maximum amplitude) after lyophilization, indicating delayed coagulation initiation and weakened clot strength. It is proposed to simultaneously supplement with fibrinogen solution and anticoagulants to balance its coagulation dynamics and bring it closer to the target MA state in normal conditions. Level C mainly showed a decrease in MA after lyophilization, suggesting impaired fibrinogen function or polymerization. It is proposed to add fibrinogen solution to restore its hypercoagulable + high fibrinogen phenotype. In addition, to ensure the protection of the overall structure of the reagents during the freeze-drying process and the stability of long-term storage, freeze-drying protectants need to be added to the formulation system as general excipients for all six levels.

[0193] 4. Screening experiment for freeze-drying protectant formulation

[0194] The screening of freeze-drying protectants is shown in Table 11 below. The solvent is purified water. The effects of different freeze-drying protectants on the product results were tested.

[0195] Table 11 Screening of Lyophilization Protectant Formulations

[0196]

[0197] Four 20ml portions of level E mixed plasma were taken and mixed with 5% lyophilization protectant A, lyophilization protectant B, lyophilization protectant C, and lyophilization protectant D respectively. After testing with a thromboelastography instrument, the mixture was dispensed into vials at 1ml each and lyophilized according to the same lyophilization procedure 3. The test data before and after lyophilization are shown in Table 12.

[0198] Table 12 Results of Thromboelastography Test after Freeze-Drying

[0199]

[0200] Table 13 Aging test results of different freeze-drying protectants

[0201]

[0202] After adding lyophilization protectants A, B, and C to the mixed plasma, the R value gradually increased and the MA value gradually decreased with the aging test time. After adding lyophilization protectant D, the R, K, Angle, and MA values ​​remained relatively stable until day 14 of the aging test, but MA showed a slight decrease at days 21 and 28. Analysis of the repeatability test results from the 28-day aging test at 37℃ (Table 13) showed that the K and MA values ​​of the quality control products lyophilized with lyophilization protectants A, B, and C all exhibited CVs > 10% to varying degrees, while the CVs of the R, K, Angle, and MA values ​​of the quality control products lyophilized with lyophilization protectant D were within 10%, with the Angle CV approaching 10%. Therefore, based on lyophilization protectant D, the formulation was gradually optimized, as shown in Table 14, with purified water as the solvent.

[0203] Table 14 Optimization of freeze-drying protectants

[0204]

[0205] Take three 20ml portions of level E mixed plasma, add 5% lyophilization protectant D1, lyophilization protectant D2, and lyophilization protectant D3 respectively, mix, and after testing with a thromboelastography instrument, dispense 1ml into each vial and lyophilize according to the same lyophilization procedure 3. The test data before and after lyophilization are shown in Table 15:

[0206] Table 15. Thromboelastography results after lyophilization with optimized freeze-dried protectant.

[0207]

[0208] Table 16. Aging test results of different optimized freeze-drying protectants

[0209]

[0210] As shown in Table 15, the 28-day aging test results of freeze-drying protectant D1 were similar to those of freeze-drying protectant D, except that MA slightly decreased after 14 days of aging. The test results of freeze-drying protectants D2 and D3 were relatively stable. Table 16 shows that the repeatability test results at 37℃ for 28 days revealed that the CVs (volume values) of the quality control samples R, K, Angle, and MA for freeze-dried samples with freeze-drying protectants D1, D2, and D3 were all less than 10%. However, the CVs of the quality control samples with freeze-drying protectants D2 and D3 were better. To save costs, freeze-drying protectant D2 (Hepes 0.1%, Mannitol 4%, PEG 5%, Trehalose 5%, Taurine 4%, HSA 2%; pH 7.4±0.2) was selected as the final freeze-drying protectant formulation. This specific combination and ratio range were discovered through extensive screening experiments. The absence of any one of these components, or changes in the ratio, would prevent the achievement of optimal technical results.

[0211] 5. Anticoagulant screening experiment

[0212] The screening of anticoagulants is shown in Table 17 below. The solvent is purified water. The effects of different anticoagulants on the product results were tested.

[0213] Table 17 Screening of Anticoagulants

[0214]

[0215] Take four 20ml portions of mixed plasma at level F, add 5% lyophilization protectant D2 to each portion, and then add 1% (w / v) anticoagulant A, anticoagulant B, anticoagulant C, and anticoagulant D from Table 17 respectively. After testing with a thromboelastography instrument, dispense 1ml into each vial and lyophilize according to the same lyophilization procedure 3. The test data before and after lyophilization are shown in Table 18.

[0216] Table 18 Aging test results after adding anticoagulant

[0217]

[0218] The accelerated stability test (37°C for 4 weeks) showed the results in Table 18. For plasma mixed with dalteparin sodium, the changes before and after lyophilization were minimal, and the 28-day aging test results were stable. For plasma mixed with heparin sodium, the R and Angle values ​​changed significantly after lyophilization. For plasma mixed with enoxaparin sodium, the R value changed significantly after lyophilization, gradually decreasing with prolonged aging test time. For plasma mixed with nadroparin sodium, the changes after lyophilization were minimal, but the R and MA values ​​changed significantly after the aging test. Therefore, dalteparin sodium has a significantly smaller impact on thromboelastographic parameters (especially R, MA, and Angle values) than heparin sodium, enoxaparin sodium, and nadroparin sodium, exhibiting lower volatility. Therefore, dalteparin sodium was chosen as the anticoagulant.

[0219] 6. Screening experiment for fibrinogen solution formulation

[0220] The screening of fibrinogen solutions is shown in Table 19 below. The solvent is purified water. The effects of different fibrinogen solutions on the product results were tested.

[0221] Table 19 Screening of Fibrinogen Solution Formulations

[0222]

[0223] Take three 20ml portions of mixed plasma at level C, add 5% lyophilization protectant D2 and 1% (w / v) anticoagulant D (1% (w / v) dalteparin sodium) to each portion respectively, then add fibrinogen solution A, fibrinogen solution B, and fibrinogen solution C respectively. After testing with a thromboelastography instrument, dispense 1ml into each vial and lyophilize according to the same procedure. The test data before and after lyophilization are shown in Table 20.

[0224] Table 20 Aging Test After Adding Fibrinogen Solution

[0225]

[0226] The experimental results showed that the quality control sample lyophilized with fibrinogen solution A showed the most significant decrease in Angle and MA values, followed by the sample lyophilized with fibrinogen solution B, while the sample lyophilized with fibrinogen solution C exhibited the best stability. Therefore, fibrinogen solution C was selected.

[0227] (iv) Plasma preparation and solution preparation

[0228] 1. Plasma preparation:

[0229] (1) Collection of anticoagulated blood: At the time of slaughter, the whole blood of fresh animals (pigs, cattle, sheep, rabbits) is mixed with 3.5% sodium citrate saline anticoagulant at a volume ratio of 9:1, and the whole blood of the anticoagulated animals is collected.

[0230] Table 21 Citric Acid Anticoagulant Formulation

[0231]

[0232] (2) Preparation of plasma: Anticoagulated animal whole blood was left to stand overnight at 4°C to allow it to separate into layers naturally. The upper plasma layer was carefully aspirated and centrifuged at 4°C and 3500 rpm for 10 min. The supernatant was collected, sodium azide was added to a final concentration of 0.05%, and the plasma was aliquoted and stored below -30°C to obtain plasma.

[0233] 2. Solution preparation:

[0234] 1% (w / v) dalteparin sodium solution: 0.1g + 9.9ml water.

[0235] 100ml (1% glycine-0.5% arginine-sodium chloride solution): 1g glycine + 0.5g arginine + 0.9g sodium chloride + 97.6ml water.

[0236] 0.5% fibrinogen solution: 50mg fibrinogen + 10ml (1% glycine-0.5% arginine-sodium chloride solution).

[0237] The freeze-drying protectant was prepared as follows: Hepes 0.1%, mannitol 4%, PEG 5%, trehalose 5%, HSA 2%, taurine 4%, and purified water as the solvent.

[0238] Example 1 Final Formula

[0239] Taking 100ml of plasma as an example, the following were added to levels A, B, C, D, E, and F: porcine plasma, bovine plasma, sheep plasma, rabbit plasma, 0.5% fibrinogen solution, 1% (W / V) dalteparin sodium solution, lyophilization protectant, and the results of thromboelastography test before lyophilization. Table 22 shows the results of the thromboelastography test before lyophilization.

[0240] Table 22 Six-Level Quality Control Product Formulation Table

[0241]

[0242] Freeze-drying was performed using the same procedure: pre-freezing at -45℃ for 6 hours; after pre-freezing, sublimation was carried out at temperature gradients of 5-10℃, with vacuum controlled at 150 ubar, holding at -40℃, -35℃, -30℃, -20℃, and -15℃ for 4 hours, 4 hours, 3 hours, 3 hours, and 3 hours respectively; vacuum controlled at 100 ubar, holding at -5℃ and 0℃ for 3 hours and 3 hours respectively; vacuum controlled at 80 ubar, holding at 5℃ and 15℃ for 3 hours and 3 hours respectively; vacuum controlled at 50 ubar, holding at 20℃ and 25℃ for 3 hours and 4 hours respectively. Test data after freeze-drying and aging are shown in Table 23.

[0243] Table 23. Aging Test Results of Level 6 Quality Control Products

[0244]

[0245]

[0246]

[0247]

[0248] The test results above show that the CV values ​​of the four parameters R, K, Angle, and MA of the six quality control products (levels A, B, C, D, E, and F) after freeze-drying and the 28-day aging test are all within 10%.

[0249] The quality control products of this invention cover six quality control levels, A to F. The coagulation parameters for each level are designed based on large-scale clinical activated coagulation test data from American Blood Technologies and Xinhua, comprehensively covering various clinical coagulation states. The core characteristics, simulated samples, corresponding clinical scenarios and symptoms for each level are shown in Table 24.

[0250] Table 24. Correspondence between core parameters of thromboelastography level 6 quality control products and clinical applications.

[0251]

[0252] Note: In the table, R represents coagulation reaction time, K represents clot formation time, Angle represents clot formation rate, and MA represents maximum clot strength. The corresponding clinical scenarios and symptoms are for reference only; actual clinical diagnosis should be performed by professional medical personnel.

[0253] Horizontal A (R value 4~9min, K value 1~4min, Angle value 55~75°, MA value 25~40mm);

[0254] Horizontal B (R value 4~10min, K value 1~4min, Angle value 55~75°, MA value 40~65mm);

[0255] Horizontal C (R value 1.5~2.5min, K value 0.8~1.0min, Angle value 72~92°, MA value 55~80mm);

[0256] Horizontal D (R value 1.0~2.5min, K value 0.5~2.2min, Angle value 60~90°, MA value 20~40mm);

[0257] Horizontal E (R value 2.5~5.0 min, K value 0.5~2.2 min, Angle value 60~90°, MA value 40~60 mm);

[0258] Horizontal F (R value > 10min, K value > 3min, Angle value < 55°, MA value < 50mm);

[0259] Its coagulation parameters (R value, K value, Angle value, MA value) range are specifically designed based on large-scale clinical activated coagulation test data (U.S. Blood Technologies, Inc., for short: Blood Technologies; the activated coagulation test reagent for normal human samples has a range of R 5~10min, K 1~3min, Angle 53~72°, MA 50~70mm; the Xinhua activated coagulation test reagent for normal human samples has a range of R 5~10min, K 1~3min, Angle 53~72°, MA 50~70mm), comprehensively covering clinical states such as hypocoagulation, normal coagulation, and hypercoagulation.

[0260] 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. Thromboelastography quality control material, characterized in that, By weight, the thromboelastography quality control material comprises: 60 parts of pig plasma, 15 parts of bovine plasma, 20 parts of sheep plasma, 15 parts of rabbit plasma, 0.02 parts of fibrinogen matrix, 0.007 parts of dalteparin sodium, and 5 parts of lyophilization protectant; The fibrinogen matrix comprises fibrinogen, glycine, and arginine; wherein the mass ratio of fibrinogen, glycine, and arginine is 0.5:1:0.

5. The freeze-drying protectant includes 4-hydroxyethylpiperazine ethanesulfonic acid, mannitol, polyethylene glycol, trehalose, taurine, and human serum albumin; wherein the mass ratio of 4-hydroxyethylpiperazine ethanesulfonic acid, mannitol, polyethylene glycol, trehalose, taurine, and human serum albumin is 0.1:4:5:5:4:

2.

2. The thromboelastography quality control product according to claim 1, characterized in that, The quality control levels of the thromboelastography quality control material are as follows: R value 4~10 min, K value 1~4 min, Angle value 55~75°, and MA value 40~65 mm.

3. The method for preparing the thromboelastography quality control sample according to claim 1 or 2, characterized in that, include: The plasma matrix, fibrinogen matrix, lyophilization protectant and anticoagulant are mixed, pre-frozen, and then sublimated at elevated temperature to obtain the final product.

4. The preparation method according to claim 3, characterized in that, The pre-freezing conditions include: the pre-freezing temperature is -45℃, and the pre-freezing time is 6 hours; The conditions for the heating and sublimation include: Under a vacuum of 150 ubar, the temperature was sequentially maintained at -40℃ for 4 h, -35℃ for 4 h, -30℃ for 3 h, -20℃ for 3 h, and -15℃ for 3 h. Under a vacuum of 100 ubar, the temperature was maintained at -5℃ for 3 h and then at 0℃ for 3 h. Under a vacuum of 80 ubar, the temperature was maintained at 5℃ for 3 h and then at 15℃ for 3 h, respectively. Under a vacuum of 50 ubar, the temperature was maintained at 20°C for 3 h and then at 25°C for 4 h.

5. The application of the thromboelastography quality control product according to claim 1 or 2, or the thromboelastography quality control product obtained by the preparation method according to claim 3 or 4, in the preparation of coagulation test products.

6. A coagulation testing product, characterized in that, This includes the thromboelastography quality control product as described in claim 1 or 2, or the thromboelastography quality control product obtained by the preparation method described in claim 3 or 4.

7. A method for detecting coagulation, characterized in that, The coagulation test product according to claim 6 is used to test the sample to be tested.

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