A blood matrix liquid D-dimer and FDP quality control product and its preparation method

Abstract

The present invention discloses a blood matrix liquid D-dimer and FDP quality control product and preparation method, including: obtaining a cross-linked fibrin clot after adding thrombin to human mixed plasma, performing a process including washing, dissolving, inactivating and ultrafiltration on the obtained cross-linked fibrin clot to obtain D-D mother liquor; obtaining a non-cross-linked fibrin polymer by adding snake venom thrombin to human mixed plasma, performing a process including washing, precipitation dissolution, inactivating and ultrafiltration on the obtained non-cross-linked fibrin clot to obtain FDP mother liquor; obtaining matrix serum by adding a buffer, a stabilizer and a preservative to human serum; respectively adding D-D mother liquor and FDP mother liquor to the matrix serum, filtering and freezing the matrix serum to which the D-D mother liquor and FDP mother liquor were added, and obtaining a D-D and FDP composite quality control product. The present invention uses human matrix serum, and the difference between different detection systems is small.

Description

Technical Field

[0001] The present invention relates to the technical field of clinical coagulation testing, and in particular to a blood matrix liquid D-dimer and FDP quality control product and a preparation method thereof. Background Art

[0002] The fibrinolytic system (abbreviated as the fibrinolytic system) is a crucial component of the body's anticoagulant function and clearance of coagulation activation products. It plays a vital role in maintaining smooth blood flow within blood vessels and tissue repair. D-dimer (DD) and fibrinogen degradation products (FDP) are the most common laboratory tests assessing the activation and function of the fibrinolytic system. The core component of the fibrinolytic system is plasminogen, which can be activated into plasmin through intrinsic, extrinsic, and exogenous activation pathways. The substrates of plasmin are fibrinogen and fibrin. Activated plasmin and plasminogen activators can be inactivated by plasminogen activator inhibitors, α2-antiplasmin, α2-macroglobulin, and other substances. When the coagulation system is activated, thrombin catalyzes the conversion of fibrinogen to fibrin and activates coagulation factor XIII to convert fibrin monomers into a cross-linked fibrin network. Simultaneously, the intrinsic activation pathway is initiated by coagulation factors XIIa, XIa, and HMWK. Activated plasmin degrades the cross-linked fibrin into fragments of various sizes, including DD, collectively referred to as FDP. When tissue-type plasminogen activator and / or urokinase-type plasminogen activator are primarily activated in the fibrinolytic system, plasminogen is activated through the extrinsic pathway, degrading fibrinogen into fragments of various sizes, also collectively referred to as FDP. However, these fragments do not contain DD, indicating that DD can only be produced through the degradation of the cross-linked fibrin network formed by the intrinsic activation pathway. However, a unique type of thrombin found in nature, snake venom-derived thrombin, can also catalyze the conversion of fibrinogen to fibrin but cannot activate coagulation factor XIII. Therefore, it cannot form a cross-linked fibrin network and, consequently, cannot produce DD. When a thrombotic disorder develops, i.e., a cross-linked fibrin clot forms within a blood vessel, rapid thrombolysis is necessary to prevent localized tissue infarction. Injections of drugs such as urokinase and streptokinase can be used to activate the fibrinolytic system through exogenous activation pathways, thereby achieving the goal of thrombolysis. Therefore, clinical laboratories use DD as a marker of thrombosis, which is extremely important for both exclusion diagnosis and thrombolytic therapy. FDP is often measured together with DD, and their clinical application value complements each other, making them crucial for the diagnosis and differential diagnosis of diseases. Accurate and reliable DD and FDP test results are the prerequisite and basis for accurate and efficient clinical diagnosis. Therefore, the use of low-cost, high-quality, easy-to-use, and appropriately concentrated composite quality control products is crucial for quality control of the testing system. Furthermore, high-performance quality control products can not only evaluate or verify measurement precision and accuracy, as well as potential analytical deviations caused by variations in reagents or analytical instruments, but can also be used for proficiency testing.

[0003] Currently, there are virtually no commercially available blood-based composite quality control products containing DD and FDP. This is primarily because the raw materials currently claimed to be DD or FDP are actually the same raw material: FDP containing DD. Extracting and purifying DD using current biotechnological methods is very costly, and the buffer background is "zero," making it easier to prepare and more stable as a quality control matrix. Therefore, quality control manufacturers typically use FDP raw material to prepare separate buffer-based DD and FDP quality control products. This technical shortcoming can lead to a number of clinical problems. First, DD and FDP are one of the most classic combinations of coagulation parameters. Using multiple, uncombined quality control products can be inconvenient for users, resulting in low quality control efficiency and delays in testing. Second, separate quality control of DD and FDP can compromise their effectiveness, leading to clinically abnormal results where DD concentrations exceed FDP concentrations. Third, the results of DD and FDP tests currently vary significantly across different testing systems, to the point of being incomparable. Using buffer as a quality control matrix differs significantly from the clinical sample matrix, leading to matrix effects that further increase the discrepancy between test results and negatively impact standardization of this test.

[0004] Clinical laboratories require quality control products to have at least two concentration levels: normal and abnormal. The normal reference range for DD is <0.5 mg / L, and the normal reference range for FDP is <5 mg / L. However, known DD and FDP composite quality control products have a DD concentration of 1-5 mg / L at level 1 and an FDP concentration of 5-15 mg / L at level 2, with even higher concentrations, which do not meet clinical requirements. Furthermore, the physiological concentration of FDP is approximately three times that of DD. Therefore, the FDP concentration in normal human clinical samples is <2 mg / L, while the detection limit of most FDP in vitro diagnostic kits is >2.5 mg / L. Therefore, preparing normal value quality control products with FDP concentrations directly from clinical samples is too low. Adding FDP raw materials containing DD makes it difficult to ensure that both DD and FDP are within the normal reference range.

[0005] In addition, most of the existing DD and FDP single or compound quality control products in this field are in the form of freeze-dried powders, which requires professional operators to use precision instruments for reconstitution, which may introduce random errors, leading to indoor quality control abnormalities and quality control product scrapping, thereby introducing great uncertainty and hidden costs. Summary of the Invention

[0006] The present invention aims to provide a method for preparing a blood-based liquid D-dimer and FDP quality control product, which is intended to address the technical problems that DD and FDP products on the market are almost all single quality control products based on buffer matrices, with very few composite quality control products and virtually no composite quality control products based on blood matrices; the technical problem that the concentrations of existing DD and FDP composite quality control products do not meet clinical requirements; and the technical problem that most existing patents / products are freeze-dried powders, which require precise reconstitution operations for use in clinical laboratories, causing inconvenience to users and potentially introducing random errors.

[0007] According to a first aspect of the present invention, the present invention provides a method for preparing a blood matrix liquid D-dimer and FDP quality control product, comprising:

[0008] The method comprises the steps of adding thrombin to human mixed plasma to obtain a cross-linked fibrin clot, washing, dissolving, inactivating and ultrafiltration the cross-linked fibrin clot to obtain a DD mother solution;

[0009] Non-cross-linked fibrin polymers are obtained by adding snake venom thrombin to human mixed plasma. The obtained non-cross-linked fibrin clots are washed, precipitated, dissolved, inactivated and ultrafiltered to obtain FDP mother liquor:

[0010] Matrix serum is obtained by adding buffers, stabilizers, and preservatives to human serum;

[0011] The DD and FDP composite quality control product is obtained by respectively adding the DD mother solution and the FDP mother solution to the matrix serum, filtering and freezing the matrix serum to which the DD mother solution and the FDP mother solution are added.

[0012] Preferably, adding the DD mother solution and the FDP mother solution to the matrix serum respectively comprises:

[0013] According to the preset concentration value of DD and FDP composite quality control product DD and the DD concentration value of matrix serum, the DD mother solution is added to the matrix serum to obtain a matrix serum solution added with the DD mother solution;

[0014] According to the preset concentration value of FDP of the DD and FDP composite quality control product and the FDP concentration value of the matrix serum solution, the FDP mother solution is added to the matrix serum solution.

[0015] Preferably, according to the preset concentration value of the control product DD and the DD concentration value of the matrix serum, adding the DD mother solution to the matrix serum comprises:

[0016] Determine the matrix serum DD concentration, and calculate the amount of the DD stock solution to be added based on the preset concentration values of DD and FDP composite quality control product DD and the determined matrix serum DD concentration value;

[0017] The DD stock solution was added to the matrix serum according to the calculated addition amount of the DD stock solution, so as to adjust the DD concentration values of the composite quality control products with different levels of DD and FDP.

[0018] Preferably, according to the preset concentration value of the quality control product FDP and the FDP concentration value of the matrix serum solution, adding the FDP mother solution to the matrix serum solution comprises:

[0019] Determine the FDP concentration of the matrix serum solution, and calculate the amount of the FDP stock solution to be added based on the preset FDP concentration value of the DD and FDP composite quality control product and the determined FDP concentration value of the matrix serum solution;

[0020] According to the calculated amount of the FDP stock solution, the FDP stock solution was added to the matrix serum solution to adjust the FDP concentration values of the DD and FDP composite quality control products at different levels.

[0021] Preferably, the method of obtaining a cross-linked fibrin clot by adding thrombin to mixed human plasma comprises:

[0022] Thrombin is added to human mixed plasma and stirred in a water bath for 10-30 minutes to allow the plasma to completely coagulate and obtain a cross-linked fibrin clot.

[0023] Preferably, washing, dissolving, inactivating and ultrafiltration of the obtained cross-linked fibrin clot comprises:

[0024] Aspirate the residual serum from the cross-linked fibrin clot and wash repeatedly with a buffer solution at pH 6.5-8.5;

[0025] The washed cross-linked fibrin clot is placed in a buffer solution, thrombolytic drugs are added, and the mixture is stirred in a water bath for 2-4 hours to dissolve the cross-linked fibrin clot;

[0026] After the cross-linked fibrin clot is dissolved, samples are taken at regular intervals to determine its DD and FDP concentrations. When the DD concentration value stabilizes, i.e., when the fibrin is completely degraded, it is placed in a 50-60°C water bath for inactivation.

[0027] The inactivated solution was ultrafiltered to remove impurities and unstable components, and the filtrate was collected, mixed, and the final DD and FDP concentrations were determined.

[0028] Preferably, the non-cross-linked fibrin polymer obtained by adding snake venom thrombin to human mixed plasma comprises:

[0029] Add snake venom thrombin to human mixed plasma and stir in a water bath for 10-30 minutes to fully activate the coagulation system and obtain non-cross-linked fibrin polymers.

[0030] Preferably, the washing, precipitation, dissolution, inactivation and ultrafiltration treatment of the obtained non-crosslinked fibrin clot comprises:

[0031] After the non-cross-linked fibrin polymer is centrifuged to obtain a precipitate, it is repeatedly washed with a buffer solution with a pH of 6.5-8.5;

[0032] The washed non-cross-linked fibrin polymer precipitate is placed in a buffer solution, a thrombolytic drug is added, and the mixture is stirred at a low speed in a water bath for 2-4 hours to dissolve the non-cross-linked fibrin polymer precipitate;

[0033] After the non-crosslinked fibrin polymer precipitate is dissolved, samples are taken at regular intervals to determine the FDP concentration. When the FDP concentration value stabilizes, i.e., when the fibrin is completely degraded, an enzyme inhibitor is added to inactivate the thrombolytic drug.

[0034] The resulting solution was ultrafiltered to remove impurities and unstable components, and the filtrate was collected, mixed, and the final FDP concentration was determined.

[0035] Preferably, the buffer added to the human serum is composed of one or two of tris(hydroxymethylaminomethane), phosphate buffer, and 4-hydroxyethylpiperazineethanesulfonic acid; the stabilizer added to the human serum is composed of one or more of sucrose, trehalose, bovine serum albumin, mannitol, and sorbitol; and the preservative added to the human serum is composed of one or more of sodium azide, potassium azide, thimerosal, Proclin 300, and Krovin 500.

[0036] According to a second aspect of the present invention, the present invention provides a blood matrix liquid D-dimer and FDP quality control product prepared according to the above method.

[0037] Compared with the prior art, the present invention has the following technical effects:

[0038] 1) Human serum is used, which has no matrix effect and small differences between different detection systems, making it more suitable as a third-party quality control product;

[0039] 2) It can save the waste of financial, material and human resources caused by the procurement, use and testing of different quality control products, facilitate clinical use and improve testing efficiency;

[0040] 3) No need to purify or purchase raw materials, low cost and easy to obtain.

[0041] 4) Concentration design better meets clinical quality control requirements;

[0042] 5) The concentrations of DD and FDP can be adjusted at will;

[0043] 6) The preparation process is easier to standardize and streamline, reducing product batch differences;

[0044] 7) DD and FDP can be combined with any coagulation test items for normal and abnormal levels simultaneously.

[0045] 8) It can avoid random errors caused by reconstitution operations, reduce personnel requirements, and facilitate use in clinical laboratories;

[0046] 9) Good performance, uniformity is better than the quality control product of lyophilized dosage form, and stability is not inferior to similar products. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 The present invention is a schematic diagram of a method for preparing a blood matrix liquid D-dimer and FDP quality control product. DETAILED DESCRIPTION

[0048] like Figure 1 As shown, the method for preparing a blood matrix liquid D-dimer and FDP composite quality control product (or simply composite quality control product) of the present invention includes:

[0049] The method comprises the steps of adding thrombin to human mixed plasma to obtain a cross-linked fibrin clot, washing, dissolving, inactivating and ultrafiltration the cross-linked fibrin clot to obtain a DD mother solution;

[0050] Non-cross-linked fibrin polymers are obtained by adding snake venom thrombin to human mixed plasma. The obtained non-cross-linked fibrin clots are washed, precipitated, dissolved, inactivated and ultrafiltered to obtain FDP mother liquor:

[0051] Matrix serum is obtained by adding buffers, stabilizers, and preservatives to human serum;

[0052] The composite quality control product is obtained by respectively adding the DD mother solution and the FDP mother solution to the matrix serum, filtering and freezing the matrix serum to which the DD mother solution and the FDP mother solution are added.

[0053] Since the composite quality control product of the present invention adopts matrix serum, there is no matrix effect, which makes the difference between different detection systems small, and has low cost, is convenient for clinical use, and improves quality control efficiency.

[0054] In addition, the present invention can add DD mother solution and FDP mother solution to the matrix serum respectively according to needs, thereby adjusting the concentrations of DD and FDP, which is more in line with clinical use needs.

[0055] The present invention can obtain cross-linked fibrin clots after adding thrombin to human mixed plasma by the following method: adding thrombin to human mixed plasma, stirring in a water bath for 10-30 minutes to completely coagulate the plasma, and obtaining cross-linked fibrin clots.

[0056] The present invention can wash, dissolve, inactivate and ultrafilter the obtained cross-linked fibrin clot by the following method: absorbing residual serum of the cross-linked fibrin clot and repeatedly washing it with a buffer solution with a pH of 6.5-8.5; placing the washed cross-linked fibrin clot in a buffer solution, adding a thrombolytic drug, and stirring in a water bath for 2-4 hours to dissolve the cross-linked fibrin clot; after the cross-linked fibrin clot is dissolved, measuring its DD and FDP concentrations at regular intervals, and after the DD concentration value stabilizes, that is, after the fibrin is completely degraded, placing it in a water bath at 50-60°C for inactivation; ultrafiltering the inactivated solution to remove impurities and unstable components, collecting the filtrate, mixing it, and measuring the final DD and FDP concentrations.

[0057] The present invention can obtain non-crosslinked fibrin polymers by adding snake venom thrombin to human mixed plasma through the following method: adding snake venom thrombin to human mixed plasma, stirring in a water bath for 10-30 minutes to fully activate the coagulation system and obtain non-crosslinked fibrin polymers.

[0058] The present invention can wash, precipitate, dissolve, inactivate and ultrafilter the obtained non-crosslinked fibrin clots by the following method: centrifuge the non-crosslinked fibrin polymer to obtain a precipitate, and then repeatedly wash it with a buffer solution with a pH of 6.5-8.5; place the washed non-crosslinked fibrin polymer precipitate in a buffer solution, add a thrombolytic drug, and stir it in a water bath at a low speed for 2-4 hours to dissolve the non-crosslinked fibrin polymer precipitate; after the non-crosslinked fibrin polymer precipitate is dissolved, sample it at regular intervals to determine the FDP concentration; after the FDP concentration value stabilizes, that is, after the fibrin is completely degraded, add an enzyme inhibitor to inactivate the thrombolytic drug; ultrafilter the obtained solution to remove impurities and unstable components, collect the filtrate, mix it, and determine the final FDP concentration.

[0059] The present invention generally adds the DD mother solution and the FDP mother solution to the matrix serum respectively, comprising the following steps: adding the DD mother solution to the matrix serum according to a preset DD concentration value of a composite quality control product and a DD concentration value of the matrix serum to obtain a matrix serum solution to which the DD mother solution is added; and adding the FDP mother solution to the matrix serum solution according to a preset FDP concentration value of a composite quality control product and a FDP concentration value of the matrix serum solution.

[0060] According to the present invention, adding the DD mother solution to the matrix serum based on the preset DD concentration value of the composite quality control product and the DD concentration value of the matrix serum generally includes the following steps: measuring the DD concentration of the matrix serum, calculating the amount of the DD mother solution to be added based on the preset DD concentration value of the composite quality control product and the measured DD concentration value of the matrix serum; and adding the DD mother solution to the matrix serum according to the calculated amount of the DD mother solution to adjust the DD concentration value of the composite quality control product at different levels.

[0061] For example, assuming the matrix serum DD concentration is a1, the composite control DD preset concentration is a2, the DD concentration of the DD mother solution is a3, and the matrix serum volume is V A , the amount or volume of DD mother liquor to be added V B :

[0062]

[0063] According to the present invention, adding the FDP mother solution to the matrix serum solution based on the preset FDP concentration value of the quality control product and the FDP concentration value of the matrix serum solution generally includes the following steps: measuring the FDP concentration of the matrix serum solution (i.e., the matrix serum to which the DD mother solution is added), calculating the amount of the FDP mother solution to be added based on the preset FDP concentration value of the composite quality control product and the measured FDP concentration value of the matrix serum solution; and adding the FDP mother solution to the matrix serum solution according to the calculated amount of the FDP mother solution to adjust the FDP concentration value of the composite quality control product at different levels.

[0064] For example, assuming the FDP concentration of the matrix serum solution is b1, the preset FDP concentration of the composite quality control is b2, the FDP concentration of the FDP mother solution is b3, and the volume of the matrix serum solution is V A+B , the amount or volume of DD mother liquor to be added V C。

[0065]

[0066] The DD preset concentration value and the FDP preset concentration value of the composite quality control product of the present invention include normal level preset concentration values and abnormal level preset concentration values.

[0067] The normal level DD preset concentration value and FDP preset concentration value of the composite quality control product of the present invention are shown in Table 1 below:

[0068] Table 1

[0069]

[0070] The abnormal level DD preset concentration value and FDP preset concentration value of the composite quality control product of the present invention are shown in Table 2 or Table 3 below:

[0071] Table 2

[0072]

[0073] Table 3

[0074]

[0075] In the present invention, the buffer added to the human serum is composed of one or two of tris(hydroxymethylaminomethane), phosphate buffer, and 4-hydroxyethylpiperazineethanesulfonic acid; the stabilizer added to the human serum is composed of one or more of sucrose, trehalose, bovine serum albumin, mannitol, and sorbitol; and the preservative added to the human serum is composed of one or more of sodium azide, potassium azide, thimerosal, Proclin 300, and Krovin 500.

[0076] More specifically, the buffer added to human serum is composed of one or two of tris(hydroxymethyl)aminomethane 0.01-1.00 mol / L, phosphate buffer 0.01-1.00 mol / L, and 4-hydroxyethylpiperazineethanesulfonic acid 0.5-30 g / L;

[0077] The stabilizer added to the human serum is composed of one or more of sucrose 20-80 g / L, trehalose 20-80 g / L, bovine serum albumin 10-50 g / L, mannitol 2-50 g / L, and sorbitol 2-50 g / L.

[0078] The preservative added to human serum consists of one or more of sodium azide 0.5-3g / L, potassium azide 0.5-3g / L, thimerosal 0.5-3g / L, Proclin 300 0.5‰~3‰, and Krovin 500 0.5‰~3‰.

[0079] On the other hand, the present invention also provides a blood matrix liquid D-dimer and FDP quality control product prepared according to the above method.

[0080] According to the method for preparing the composite quality control product of the present invention, the present invention first prepares DD mother liquor and FDP mother liquor, and then uses the DD mother liquor and FDP mother liquor to prepare the composite quality control product. The following example specifically illustrates the preparation process of the composite quality control product of the present invention:

[0081] (1) Preparation of DD mother liquor

[0082] ① Take human mixed plasma, add thrombin at a rate of 1-20 U / mL, and stir in a 37°C water bath for 10-30 minutes to allow the plasma to completely coagulate and obtain a cross-linked fibrin clot;

[0083] ② Remove the clot and drain the residual serum, then wash it 2-3 times with a buffer solution with a pH of 6.5-8.5, including but not limited to PBS buffer, Tris buffer, and HEPES buffer;

[0084] ③ Place the washed fibrin clot in a small amount of buffer, add thrombolytic drugs at a rate of 100-2000 U / mL, and stir in a 37°C water bath for 2-4 hours. Thrombolytic drugs include but are not limited to urokinase, streptokinase, staphylokinase, alteplase, reteplase, pamiplase, tenecteplase, etc.;

[0085] ④ After the fibrin clot is dissolved, samples are taken every 30 minutes to measure the DD and FDP concentrations. When the DD concentration value stabilizes, i.e., when the fibrin is completely degraded, the sample is placed in a 56°C water bath to inactivate it.

[0086] ⑤ Filter the inactivated solution through a 0.22 μm filter membrane and then ultrafilter again to remove impurities and unstable components. Collect the filtrate, mix it, and determine the final DD and FDP concentrations.

[0087] (2) Preparation of FDP mother liquor

[0088] ① Take human mixed plasma, add snake venom thrombin at a rate of 1-20 U / mL, and stir in a 37°C water bath for 10-30 minutes to fully activate the coagulation system and obtain non-cross-linked fibrin aggregates;

[0089] ② The polymer is centrifuged and washed with a buffer having a pH of 6.5-8.5, repeated 2-3 times, wherein the buffer includes but is not limited to PBS buffer, Tris buffer, and HEPES buffer;

[0090] ③ Place the washed precipitate in a small amount of buffer, add thrombolytic drugs at a rate of 100-2000 U / mL, and stir at low speed in a 37°C water bath for 2-4 hours. Thrombolytic drugs include but are not limited to urokinase, streptokinase, staphylokinase, alteplase, reteplase, pamiplase, tenecteplase, etc.;

[0091] ④ After the precipitate is dissolved, samples are taken every 30 minutes to measure the FDP concentration. After the FDP concentration stabilizes, i.e., after the fibrin is completely degraded, enzyme inhibitors are added to inactivate the thrombolytic drugs, including but not limited to one or more of aprotinin, plasminogen activator inhibitor-1, α2-antiplasmin, aminomethylbenzoic acid, tranexamic acid, and glycine;

[0092] ⑤ The resulting solution was filtered through a 0.22 μm filter membrane and then ultrafiltered again to remove impurities and unstable components. The filtrate was collected, mixed, and the final FDP concentration was determined.

[0093] (3) Preparation of composite quality control products

[0094] ① Filter human serum through a 0.45 μm filter membrane;

[0095] ② Adding a buffer, a stabilizer, and a preservative to obtain matrix serum, wherein the buffer is composed of one or two of tris(hydroxymethyl)aminomethane 0.01-1.00 mol / L, phosphate buffer 0.01-1.00 mol / L, and 4-hydroxyethylpiperazineethanesulfonic acid 0.5-30 g / L; the stabilizer is composed of one or more of sucrose 20-80 g / L, trehalose 20-80 g / L, bovine serum albumin 10-50 g / L, mannitol 2-50 g / L, and sorbitol 2-50 g / L; and the preservative is composed of one or more of sodium azide 0.5-3 g / L, potassium azide 0.5-3 g / L, thimerosal 0.5-3 g / L, Proclin 300 0.5‰-3‰, and Krovin 500 0.5‰-3‰;

[0096] ③ Determine the matrix serum DD concentration;

[0097] ④D-D concentration adjustment: Calculate the amount of DD stock solution added based on the matrix serum measurement value and the preset DD concentration of the quality control product and add it, so as to adjust the DD of the quality control products of different levels to fall within the preset concentration value range shown in Table 1, 2 or 3 below;

[0098] ⑤ Determine the FDP concentration of the solution obtained in step ④;

[0099] ⑥FDP concentration adjustment: Calculate the amount of FDP stock solution added based on the solution measurement value obtained in step ④ and the preset FDP concentration of the quality control product and add it, so as to adjust the FDP of the quality control product of different levels to fall within the preset concentration value range shown in Table 1, 2 or 3 below;

[0100] ⑦ The solution obtained in step ⑥ was filtered through a 0.22 μm filter membrane, mixed, and stored stably at -20°C or below after aliquoting.

[0101] Table 1

[0102]

[0103] or Table 2

[0104]

[0105] or Table 3

[0106]

[0107] Example 1: Preparation of normal level quality control product

[0108] (1) Preparation of DD mother solution: 30 mL of human mixed plasma was taken, 10 U / mL of thrombin was added, and the mixture was stirred in a 37°C water bath for 30 min to completely coagulate the plasma and obtain a cross-linked fibrin clot; the clot was removed and the residual serum was aspirated, and the mixture was washed three times with PBS buffer at pH 7.4; the washed fibrin clot was placed in 5 mL of buffer, urokinase was added at a concentration of 1000 U / mL, and the mixture was stirred in a 37°C water bath for 2 hours. During this period, samples were taken every 30 minutes to measure the DD and FDP concentrations. After the DD concentration value was basically stable, that is, the fibrin was completely degraded, the mixture was placed in a 56°C water bath for inactivation; the inactivated solution was filtered with a 0.22 μm filter membrane and then ultrafiltered again, the filtrate was collected and mixed, and the final DD and FDP concentrations were measured. The specific results are shown in Table 4:

[0109] Table 4

[0110]

[0111] (2) Preparation of FDP mother solution: 30 mL of human mixed plasma was added with 10 U / mL of snake venom thrombin, and stirred in a 37°C water bath for 30 min to obtain non-cross-linked fibrin polymers; the polymer was centrifuged and washed with PBS buffer at pH 7.4, and repeated three times. 5 mL of buffer was added to the final precipitate, and urokinase was added at a concentration of 1000 U / mL. The mixture was stirred in a 37°C water bath for 2 hours. During this period, samples were taken every 30 minutes to measure the FDP concentration. After the FDP concentration value stabilized, that is, when the fibrin was completely degraded, α2-antiplasmin (5 U / mL) and plasminogen activator inhibitor-1 (100 U / mL) were added to inactivate the activated plasmin and the added urokinase. After stirring in a 37°C water bath for 30 min, the solution was filtered with a 0.22 μm filter membrane and then ultrafiltered again. The filtrate was collected and mixed, and the final FDP concentration was measured. The specific results are shown in Table 5:

[0112] Table 5

[0113]

[0114] (3) Preparation of DD and FDP composite quality control products

[0115] ① Take 1 L of human serum and filter it through a 0.45 μm filter membrane to obtain filtered serum;

[0116] ② Add buffer, stabilizer and preservative to the filtered serum according to Table 6 to obtain matrix serum:

[0117] Table 6

[0118]

[0119] ③ The matrix serum DD concentration was determined to be 0.24 mg / L;

[0120] ④ Add 53 μL of DD stock solution according to the matrix serum measurement value and the preset concentration of quality control product DD (0.4 mg / L);

[0121] ⑤ The FDP concentration of the solution obtained in step ④ was 1.2 mg / L;

[0122] ⑥ According to the solution measured in step ④ and the preset concentration of the quality control FDP (3.5 mg / L), add 434 μL of FDP stock solution;

[0123] ⑦ The solution obtained in step ⑥ was filtered through a 0.22 μm filter membrane and mixed to obtain a normal level quality control product;

[0124] ⑧The final concentrations of the quality control substances DD and FDP were 0.38 mg / L and 3.6 mg / L respectively;

[0125] ⑨Mix well, divide into aliquots and store at -20℃ or below.

[0126] Example 2: Preparation of Abnormal Level Quality Control

[0127] The difference from Example 1 is that the preset concentration of DD is 1.5 mg / L and the preset concentration of FDP is 10 mg / L. Based on this, the amounts of DD stock solution and FDP stock solution added are calculated to be 420 μL and 1245 μL, respectively. The obtained concentrations of DD and FDP in the control products are 1.46 mg / L and 9.7 mg / L, respectively. Other conditions are the same.

[0128] Comparative Example 1: Preparation of normal level quality control product

[0129] The difference from Example 1 is that the preparation volume was 10 mL, and only homemade DD stock solution or purchased FDP raw material (FDP concentration of 1.21 mg / mL) was added. When only homemade DD stock solution was added, when the DD concentration was within the normal reference range, the FDP concentration was below the lower limit of linearity of the kit, failing to guarantee the precision and quality control effect of the quality control sample. When the addition amount was increased to meet the required FDP concentration, the DD concentration exceeded the normal reference range. The same situation occurred when only purchased FDP raw material was added, indicating that the raw material contained DD. Specific results are shown in Table 7:

[0130] Table 7

[0131]

[0132] The results showed that Examples 1 and 2 were able to achieve DD and FDP concentrations that simultaneously met the requirements for normal or abnormal quality control levels; whereas Comparative Example 1 was unable to prepare a normal-level quality control product with DD and FDP concentrations both within the normal reference range.

[0133] Uniformity test of quality control products in Example 1 and Example 2

[0134] The examples 1 and 2 of the present invention and the commercially available quality control products (normal DD quality control product, abnormal DD quality control product, normal FDP quality control product, abnormal FDP quality control product, all of which are freeze-dried formulations with buffer matrix) were reconstituted or accurately reconstituted with the indicated volume of distilled water, and the homogeneity test was performed according to the following method: 10 minimum packaging units of quality control products were randomly selected for each quality control product and randomly numbered 1 to 10, and each packaging unit was measured 3 times. Considering the random variation of the measurement system caused by factors such as time, the 3 measurements were performed in different orders, such as 1, 3, 5, 7, 9, 2, 4, 6, 8, 10, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 2, 4, 6, 8, 10, 1, 3, 5, 7, 9. Calculate according to formulas (1) to (11): F 、 S bb 、 S r and CV 瓶间 , the calculation results are shown in Table 8

[0135] …………………Formula (1)

[0136] …………………………Formula (2)

[0137] …………………Formula (3)

[0138] ………………………………………… Formula (4)

[0139] …………………………………………Formula (5)

[0140] ……………Formula (6)

[0141] ……………………Formula (7)

[0142] ……………Formula (8)

[0143] ………………………………Formula (9)

[0144] ……………………………………Formula (10)

[0145] ………………………………………Formula (11)

[0146] Where:

[0147] SS 总和 、 SS 瓶间 、 SS 瓶内 —are the sum of variances, between-bottle variance, and within-bottle variance, respectively;

[0148] MS 瓶间 、 MS 瓶内 —Inter-bottle mean square, intra-bottle mean square;

[0149] x i —Specify the parameter’s i-th measurement value or calculation result;

[0150] — overall mean value;

[0151] n i —Number of repeated measurements of sample i;

[0152] x ij —the jth result of sample i;

[0153] — degrees of freedom;

[0154] F — F Test value;

[0155] n 0—valid measurement times;

[0156] α—the number of samples drawn;

[0157] N — total number of tests;

[0158] S bb —Inter-bottle standard deviation;

[0159] S r —Within-bottle standard deviation (repeatability standard deviation).

[0160] Table 8

[0161]

[0162] Calculate the test data and look up the table to know that: , , , F 0.05(v1,v2) =2.3928, all quality control products DD and FDP F Average value < F 0.05(v1,v2) , indicating good uniformity between bottles, while the DD and FDP of Example 1 and Example 2 CV 瓶间 All < the corresponding items of commercially available quality control products CV 瓶间 , proving that the repeatability and precision of the quality control product of the present invention are superior to those of commercially available quality control products.

[0163] 6.6 Opening stability test of quality control products in Example 1 and Example 2

[0164] The Example 1 and Example 2 of the present invention and the commercially available quality control products (normal DD quality control product, abnormal DD quality control product, normal FDP quality control product, abnormal FDP quality control product, all of which are freeze-dried formulations of buffer matrix) were reconstituted or accurately reconstituted with distilled water according to the labeled volume, stored at 2°C-8°C, and samples were taken every 1 day, 3 days, 5 days, 7 days, 9 days, 11 days, and 14 days to detect DD and FDP. t The test table is used to test the significance of the slope trend. For a 95% confidence level, when b 1∣ <t 0.05,n-2 ×s( b 1) indicates that the stability results of the quality control product have no significant trend in change; otherwise, the stability results have a significant trend in change. The test results are shown in Tables 9 and 10:

[0165] Table 9 t Inspection Form

[0166]

[0167] Table 10

[0168]

[0169] The test data were subjected to trend test analysis according to the t-test table. The results showed that the DD and FDP composite quality control products prepared by the present invention were stored at 2°C-8°C for 9 days after reconstitution, and there was no significant change trend in DD and FDP. However, the normal value and abnormal value quality control products from commercial sources, evaluated under the same conditions after reconstitution, showed that both DD and FDP showed a decreasing trend, which fully proves that the quality control products of the present invention are more stable than commercially available quality control products.

[0170] Although the present invention has been described in detail above, it is not limited thereto, and those skilled in the art can make various modifications based on the principles of the present invention. Therefore, any modifications made based on the principles of the present invention should be understood to fall within the scope of protection of the present invention.

Claims

1. A method for preparing a blood matrix liquid D-dimer and FDP quality control product, comprising: The method comprises the steps of adding thrombin to human mixed plasma to obtain a cross-linked fibrin clot, and performing a treatment including washing, dissolving, inactivating and ultrafiltration on the cross-linked fibrin clot to obtain a DD mother solution; Non-crosslinked fibrin polymers are obtained by adding snake venom thrombin to human mixed plasma. The obtained non-crosslinked fibrin clots are subjected to treatments including washing, precipitation, dissolution, inactivation and ultrafiltration to obtain FDP mother solution: Matrix serum is obtained by adding buffers, stabilizers, and preservatives to human serum; The DD and FDP composite quality control product is obtained by respectively adding the DD mother solution and the FDP mother solution to the matrix serum, filtering and freezing the matrix serum to which the DD mother solution and the FDP mother solution are added.

2. The preparation method according to claim 1, wherein adding the DD mother solution and the FDP mother solution to the matrix serum comprises: According to the preset concentration value of DD and FDP composite quality control product DD and the DD concentration value of matrix serum, the DD mother solution is added to the matrix serum to obtain a matrix serum solution added with the DD mother solution; According to the preset concentration value of FDP of the DD and FDP composite quality control product and the FDP concentration value of the matrix serum solution, the FDP mother solution is added to the matrix serum solution.

3. The preparation method according to claim 2, wherein the DD mother solution is added to the matrix serum according to the preset DD concentration value of the quality control product and the DD concentration value of the matrix serum, comprising: Determine the matrix serum DD concentration, and calculate the amount of the DD stock solution to be added based on the preset concentration values of DD and FDP composite quality control product DD and the determined matrix serum DD concentration value; The DD stock solution was added to the matrix serum according to the calculated addition amount of the DD stock solution, so as to adjust the DD concentration values of the composite quality control products with different levels of DD and FDP.

4. The preparation method according to claim 2, wherein the FDP mother solution is added to the matrix serum solution according to the preset concentration value of the quality control FDP and the FDP concentration value of the matrix serum solution, comprising: Determine the FDP concentration of the matrix serum solution, and calculate the amount of the FDP stock solution to be added based on the preset FDP concentration value of the DD and FDP composite quality control product and the determined FDP concentration value of the matrix serum solution; According to the calculated amount of the FDP stock solution, the FDP stock solution was added to the matrix serum solution to adjust the FDP concentration values of the composite quality control products with different levels of DD and FDP.

5. The preparation method according to claim 1, wherein the step of adding thrombin to mixed human plasma to obtain a cross-linked fibrin clot comprises: Thrombin is added to human mixed plasma and stirred in a water bath for 10-30 minutes to allow the plasma to completely coagulate and obtain a cross-linked fibrin clot.

6. The preparation method according to claim 5, wherein the steps of washing, dissolving, inactivating and ultrafiltration of the cross-linked fibrin clot obtained comprise: Aspirate the residual serum from the cross-linked fibrin clot and wash repeatedly with a buffer solution at pH 6.5-8.5; The washed cross-linked fibrin clot is placed in a buffer solution, thrombolytic drugs are added, and the mixture is stirred in a water bath for 2-4 hours to dissolve the cross-linked fibrin clot; After the cross-linked fibrin clot is dissolved, samples are taken at regular intervals to measure the DD concentration. When the DD concentration value stabilizes, i.e., when the fibrin is completely degraded, it is placed in a 50-60°C water bath to inactivate it. Then ultrafiltration was performed to remove impurities and unstable components, and the filtrate was collected, mixed, and the final DD and FDP concentrations were determined.

7. The preparation method according to claim 1, wherein the non-crosslinked fibrin polymer is obtained by adding snake venom thrombin to human mixed plasma, comprising: Add snake venom thrombin to human mixed plasma and stir in a water bath for 10-30 minutes to fully activate the coagulation system and obtain non-cross-linked fibrin polymers.

8. The preparation method according to claim 7, wherein the treatment of the obtained non-crosslinked fibrin clot including washing, precipitation, dissolution, inactivation and ultrafiltration comprises: After the non-cross-linked fibrin polymer is centrifuged to obtain a precipitate, it is repeatedly washed with a buffer solution with a pH of 6.5-8.5; The washed non-cross-linked fibrin polymer precipitate is placed in a buffer solution, a thrombolytic drug is added, and the mixture is stirred at a low speed in a water bath for 2-4 hours to dissolve the non-cross-linked fibrin polymer; After the non-crosslinked fibrin polymer is dissolved, samples are taken at regular intervals to determine the FDP concentration. When the FDP concentration value stabilizes, i.e., when the fibrin is completely degraded, an enzyme inhibitor is added to inactivate the thrombolytic drug. Then ultrafiltration was performed to remove impurities and unstable components, and the filtrate was collected, mixed, and the final FDP concentration was determined.

9. The preparation method according to claim 1, wherein the buffer added to the human serum is composed of one or two of tris(hydroxymethylaminomethane), phosphate buffer, and 4-hydroxyethylpiperazineethanesulfonic acid; the stabilizer added to the human serum is composed of one or more of sucrose, trehalose, bovine serum albumin, mannitol, and sorbitol; and the preservative added to the human serum is composed of one or more of sodium azide, potassium azide, thimerosal, Proclin 300, and Krovin 500.

10. A blood matrix liquid D-dimer and FDP quality control product prepared according to the method according to any one of claims 1 to 9.

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