Blood matrix liquid D-dimer, FDP quality control product and preparation method

Abstract

The invention discloses a blood matrix liquid D-dimer, an FDP quality control product and a preparation method, and the preparation method comprises the following steps: adding thrombin into human mixed plasma to obtain a cross-linked fibrin clot, and carrying out washing, dissolving, inactivation and ultrafiltration treatment on the obtained cross-linked fibrin clot to obtain a D-D mother solution; the preparation method comprises the following steps: adding snake venom thrombin into human mixed plasma to obtain a non-crosslinked fibrin polymer, and carrying out washing, precipitation dissolution, inactivation and ultrafiltration treatment on the obtained non-crosslinked fibrin clot to obtain FDP mother liquor; a buffer agent, a stabilizer and a preservative are added into human serum to obtain matrix serum; the D-D mother liquor and the FDP mother liquor are respectively added into matrix serum, and the matrix serum added with the D-D mother liquor and the FDP mother liquor is filtered and frozen to obtain the D-D and FDP composite quality control product. The human matrix serum is adopted, and the difference between different detection systems is small.

Description

Technical Field

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

[0002] The fibrinolytic system (abbreviated as the fibrinolysis system) is an important part of the body's anticoagulation and clearance of coagulation activation products, and plays an important role in maintaining blood flow in blood vessels and tissue repair. D-dimer (D-D) and fibrin(ogen) degradation products (FDP) are the most common laboratory test items for evaluating the activation and function of the body's fibrinolysis system. The core component of the fibrinolysis system is plasminogen, which can be activated into plasmin through the internal activation pathway, external activation pathway, and exogenous activation pathway. The substrates of plasmin are fibrinogen and fibrin. Activated plasmin and plasminogen activators can be inhibited by plasminogen activator inhibitor, α 2 -antiplasmin, α 2- Inactivation of α2-macroglobulin, etc. When the coagulation system is activated, thrombin catalyzes the conversion of fibrinogen into fibrin and activates factor XIII to convert fibrin monomers into cross-linked reticular fibrin. At the same time, the intrinsic activation pathway is initiated through factor XIIa, factor XIa, HMWK, etc. Activated plasmin degrades cross-linked fibrin into fragments of various sizes, including D-D. These fragments are collectively called FDP. When tissue-type plasminogen activator and / or urokinase-type plasminogen activator in the fibrinolytic system are primarily activated, plasminogen is activated through the extrinsic activation pathway, and fibrinogen is degraded into fragments of various sizes. These fragments are also collectively called FDP, but they do not contain D-D. It can be seen that D-D can only be produced by the degradation of cross-linked reticular fibrin formed through the intrinsic activation pathway. However, there is a special thrombin in nature, namely snake venom thrombin. This type of thrombin can also catalyze the conversion of fibrinogen into fibrin, but it cannot activate factor XIII, so cross-linked reticular fibrin cannot be formed, and naturally D-D cannot be produced. When a thrombotic disease occurs in the body, that is, a cross-linked fibrin clot forms in the blood vessel, in order to avoid local tissue infarction, thrombolysis needs to be carried out as soon as possible. At this time, drugs such as urokinase and streptokinase can be injected to activate the fibrinolytic system through the exogenous activation pathway, so as to achieve the purpose of thrombolytic therapy. Therefore, clinical laboratories use D-D as a marker of thrombosis, which has extremely important clinical significance in differential diagnosis and thrombolytic therapy. At the same time, FDP is often detected together with D-D, and its clinical application value has a complementary effect, which is crucial for the diagnosis and differential diagnosis of diseases. The accuracy and reliability of the D-D and FDP test results are the premise and basis for clinicians to make correct and efficient judgments on diseases. Therefore, it is extremely important to use a composite quality control product with low cost, good quality, convenient operation and appropriate concentration to carry out the quality control of the detection system. At the same time, a quality control product with good performance can not only be used to evaluate or verify the precision of measurement, the accuracy of measurement, and the analysis deviation that may occur in the detection system due to changes in reagents or analytical instruments, but also be used for proficiency testing.

[0003] At present, there are basically no commercial quality control products that combine D-D and FDP in blood matrix on the market. The main reason is that the so-called D-D or FDP raw materials are actually the same raw material, that is, the FDP raw material containing D-D. The cost of extracting and purifying D-D by current biotechnological means is very high, and the buffer background is "0", which is easier to prepare as the quality control product matrix and has better stability. Therefore, quality control product manufacturers usually use FDP raw materials to prepare single-item quality control products of D-D and FDP in buffer matrix respectively. This technical defect will lead to a series of clinical problems. First, D-D and FDP are one of the most classic item combinations in coagulation tests. Using multiple non-combined quality control products will bring inconvenience to users, resulting in low quality control efficiency and delaying the detection work. Second, separate quality control of D-D and FDP may affect the quality control effect, resulting in abnormal results where the D-D concentration > FDP concentration clinically. Third, the detection results of D-D and FDP items currently vary greatly among different detection systems, and are even incomparable. Using buffer as the quality control product matrix has too large a difference from the clinical sample matrix, resulting in matrix effects, which will further expand the differences between detection results and have a negative impact on the standardization of this item.

[0004] Clinical laboratories require that quality control products have at least two concentration levels, normal and abnormal. The normal reference range of D-D is < 0.5 mg / L, and the normal reference range of FDP is < 5 mg / L. The D-D concentration of the known combined quality control product of D-D and FDP at level 1 is 1 - 5 mg / L, and the FDP concentration is 5 - 15 mg / L. The concentration at level 2 is even higher, which does not meet the clinical requirements. Moreover, the physiological concentration of FDP is about 3 times that of D-D. Therefore, the FDP concentration in normal human clinical samples is < 2 mg / L, while the detection lower limit of most FDP in vitro diagnostic kits is > 2.5 mg / L. So, directly using clinical samples to prepare the quality control product with normal value results in too low FDP concentration, and adding the FDP raw material containing D-D makes it difficult to ensure that both D-D and FDP are within the normal reference range.

[0005] In addition, most of the existing single-item or combined quality control products of D-D and FDP in this field adopt the freeze-dried powder dosage form, which requires professional operators to use precision instruments for reconstitution. This may introduce random errors, resulting in abnormal in-house quality control and scrapping of quality control products, thus introducing great uncertainty and hidden costs. Summary of the Invention

[0006] The object of the present invention is to provide a preparation method of a blood matrix liquid D-dimer and FDP quality control product, which is used to solve the technical problems that almost all D-D and FDP products on the market are single-item quality control products with a buffer matrix, there are very few composite quality control products, and there are basically no composite quality control products with a blood matrix; the concentration of existing D-D and FDP composite quality control products does not meet clinical requirements; and most of the existing patent / product dosage forms use freeze-dried powder, which requires precise reconstitution operations when used in clinical laboratories, thus causing inconvenience to users and possibly introducing random errors.

[0007] According to the first aspect of the present invention, the present invention provides a preparation method of a blood matrix liquid D-dimer and FDP quality control product, including: Adding thrombin to human mixed plasma to obtain cross-linked fibrin clots, washing, dissolving, inactivating, and ultrafiltering the obtained cross-linked fibrin clots to obtain a D-D mother liquor; Adding snake venom-like thrombin to human mixed plasma to obtain non-cross-linked fibrin polymers, washing, precipitating and dissolving, inactivating, and ultrafiltering the obtained non-cross-linked fibrin clots to obtain an FDP mother liquor: Adding a buffer, a stabilizer, and a preservative to human serum to obtain a matrix serum; Adding the D-D mother liquor and the FDP mother liquor to the matrix serum respectively, filtering and freezing the matrix serum added with the D-D mother liquor and the FDP mother liquor to obtain a D-D and FDP composite quality control product.

[0008] Preferably, adding the D-D mother liquor and the FDP mother liquor to the matrix serum respectively includes: According to the D-D preset concentration value of the D-D and FDP composite quality control product and the D-D concentration value of the matrix serum, adding the D-D mother liquor to the matrix serum to obtain a matrix serum solution added with the D-D mother liquor; According to the FDP preset concentration value of the D-D and FDP composite quality control product and the FDP concentration value of the matrix serum solution, adding the FDP mother liquor to the matrix serum solution.

[0009] Preferably, adding the D-D mother liquor to the matrix serum according to the D-D preset concentration value of the quality control product and the D-D concentration value of the matrix serum includes: Measuring the D-D concentration of the matrix serum, and calculating the addition amount of the D-D mother liquor according to the D-D preset concentration value of the D-D and FDP composite quality control product and the measured D-D concentration value of the matrix serum; Adding the D-D mother liquor to the matrix serum according to the calculated addition amount of the D-D mother liquor to adjust the D-D concentration value of different levels of D-D and FDP composite quality control products.

[0010] Preferably, adding the FDP stock solution to the matrix serum solution according to the preset concentration value of FDP in the quality control product and the FDP concentration value of the matrix serum solution includes: Measuring the FDP concentration of the matrix serum solution, and calculating the addition amount of the FDP stock solution according to the preset concentration value of FDP in the D-D and FDP composite quality control product and the measured FDP concentration value of the matrix serum solution; Adding the FDP stock solution to the matrix serum solution according to the calculated addition amount of the FDP stock solution, so as to adjust the FDP concentration values of the D-D and FDP composite quality control products at different levels.

[0011] Preferably, obtaining cross-linked fibrin clots by adding thrombin to human mixed plasma includes: Adding thrombin to human mixed plasma, and performing water bath stirring for 10 - 30 min to completely solidify the plasma, thereby obtaining cross-linked fibrin clots.

[0012] Preferably, washing, dissolving, inactivating, and ultrafiltrating the obtained cross-linked fibrin clots includes: Absorbing the residual serum of the cross-linked fibrin clots, and repeatedly washing with a buffer solution having a pH of 6.5 - 8.5; Placing the washed cross-linked fibrin clots in a buffer solution, adding a thrombolytic drug, and performing water bath stirring for 2 - 4 hours to dissolve the cross-linked fibrin clots; After the cross-linked fibrin clots are dissolved, sampling at regular intervals to measure their D-D and FDP concentrations. After the D-D concentration value is stable, that is, after the fibrin is completely degraded, inactivating it in a water bath at 50 - 60 °C; Ultrafiltering the inactivated solution to remove impurities and unstable components, collecting the filtrate, mixing it evenly, and measuring the final D-D and FDP concentrations.

[0013] Preferably, non-cross-linked fibrin polymers obtained by adding snake venom-like thrombin to human mixed plasma include: Adding snake venom-like thrombin to human mixed plasma, and performing water bath stirring for 10 - 30 min to fully activate the coagulation system, thereby obtaining non-cross-linked fibrin polymers.

[0014] Preferably, washing, precipitating and dissolving, inactivating, and ultrafiltrating the obtained non-cross-linked fibrin clots includes: After centrifuging the non-cross-linked fibrin polymers to obtain precipitates, repeatedly washing with a buffer solution having a pH of 6.5 - 8.5; Placing the washed precipitate of the non-cross-linked fibrin polymers in a buffer solution, adding a thrombolytic drug, and performing low-speed water bath stirring for 2 - 4 hours to dissolve the precipitate of the non-cross-linked fibrin polymers; After the non-crosslinked fibrin polymer precipitate is dissolved, samples are taken at regular intervals to measure the FDP concentration. After the FDP concentration value stabilizes, that is, when the fibrin is completely degraded, an enzyme inhibitor is added to inactivate the thrombolytic drug. The resulting solution is ultrafiltered to remove impurities and unstable components, and the filtrate is collected, mixed, and the final FDP concentration is measured.

[0015] Preferably, the buffer added to human serum is composed of one or two of tris(hydroxymethyl)aminomethane, phosphate buffer, and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; the stabilizer added to human serum is composed of one or several of sucrose, trehalose, bovine serum albumin, mannitol, and sorbitol; the preservative added to human serum is composed of one or several of sodium azide, potassium azide, thimerosal, Proclin 300, and Krovin 500.

[0016] According to the second aspect of the present invention, the present invention provides a blood matrix liquid D-dimer and FDP quality control product prepared by the above method.

[0017] Compared with the prior art, the present invention has the following technical effects: 1) Using human matrix serum, there is no matrix effect, and the difference between different detection systems is small, which is more suitable as a third-party quality control product; 2) It can save the waste of financial, material, and human resources caused by the procurement, use, and detection of different quality control products, facilitate clinical use, and improve the detection work efficiency; 3) There is no need for purification and raw material purchase, the cost is low, and it is easy to obtain.

[0018] 4) The concentration design better meets the clinical quality control requirements; 5) Arbitrary concentration adjustment of D-D and FDP can be achieved; 6) The preparation process is easier to standardize and process, reducing the batch-to-batch difference of products; 7) Arbitrary normal-level and abnormal-level combinations of D-D and FDP with any coagulation test item can be achieved.

[0019] 8) It can avoid the random error caused by the reconstitution operation, reduce the personnel requirements, and facilitate the use in clinical laboratories; 9) It has good performance, the uniformity is better than that of the freeze-dried quality control product, and the stability is not inferior to that of the same type of product. Brief Description of the Drawings

[0020] Figure 1 It is a schematic diagram of the method for preparing a blood matrix liquid D-dimer and FDP quality control product of the present invention. Detailed Embodiments

[0021] As Figure 1As shown in the figure, a preparation method of a blood matrix liquid D-dimer and FDP composite quality control product (or simply referred to as the composite quality control product) of the present invention includes: After adding thrombin to human mixed plasma, cross-linked fibrin clots are obtained. The obtained cross-linked fibrin clots are washed, dissolved, inactivated, and ultrafiltered to obtain a D-D mother liquor; After adding snake venom-like thrombin to human mixed plasma, non-cross-linked fibrin polymers are obtained. The obtained non-cross-linked fibrin clots are washed, precipitated and dissolved, inactivated, and ultrafiltered to obtain an FDP mother liquor: Matrix serum is obtained by adding a buffer, a stabilizer, and a preservative to human serum; By separately adding the D-D mother liquor and the FDP mother liquor to the matrix serum, and filtering and freezing the matrix serum added with the D-D mother liquor and the FDP mother liquor, a composite quality control product is obtained.

[0022] Since the composite quality control product of the present invention uses matrix serum, there is no matrix effect, resulting in small differences between different detection systems, low cost, convenient for clinical use, and improved quality control efficiency.

[0023] In addition, the present invention can separately add the D-D mother liquor and the FDP mother liquor to the matrix serum according to needs, so as to realize the adjustment of the D-D and FDP concentrations, which better meets the clinical use requirements.

[0024] The present invention can obtain cross-linked fibrin clots by the following method: adding thrombin to human mixed plasma and stirring in a water bath for 10 - 30 min to completely coagulate the plasma to obtain cross-linked fibrin clots.

[0025] The present invention can wash, dissolve, inactivate, and ultrafilter the obtained cross-linked fibrin clots by the following method: sucking dry the residual serum of the cross-linked fibrin clots and repeatedly washing with a buffer solution with a pH of 6.5 - 8.5; placing the washed cross-linked fibrin clots in a buffer solution, adding a thrombolytic drug, and stirring in a water bath for 2 - 4 hours to dissolve the cross-linked fibrin clots; after the cross-linked fibrin clots are dissolved, measure their D-D and FDP concentrations at regular intervals. After the D-D concentration value is stable, that is, after the fibrin is completely degraded, inactivate it in a water bath at 50 - 60 °C; ultrafilter the inactivated solution to remove impurities and unstable components, collect the filtrate, mix it evenly, and measure the final D-D and FDP concentrations.

[0026] The present invention can obtain non-cross-linked fibrin polymers by the following method: adding snake venom-like thrombin to human mixed plasma and stirring in a water bath for 10 - 30 min to fully activate the coagulation system to obtain non-cross-linked fibrin polymers.

[0027] The non-crosslinked fibrin clot obtained in the present invention can be washed, precipitated and dissolved, inactivated, and ultrafiltered by the following methods: after centrifuging the non-crosslinked fibrin polymer to obtain a precipitate, it is repeatedly washed with a buffer solution having a pH of 6.5 - 8.5; the precipitate of the washed non-crosslinked fibrin polymer is placed in a buffer solution, a thrombolytic drug is added, and it is stirred at a low speed in a water bath for 2 - 4 hours to dissolve the precipitate of the non-crosslinked fibrin polymer; after the precipitate of the non-crosslinked fibrin polymer is dissolved, samples are taken at regular intervals to measure the FDP concentration, and after the FDP concentration value is stable, that is, when the fibrin is completely degraded, an enzyme inhibitor is added thereto to inactivate the thrombolytic drug; the obtained solution is ultrafiltered to remove impurities and unstable components, and the filtrate is collected, mixed, and the final FDP concentration is measured.

[0028] The steps of adding the D-D stock solution and the FDP stock solution to the matrix serum in the present invention generally include: according to the preset concentration value of the D-D in the composite quality control product and the D-D concentration value in the matrix serum, adding the D-D stock solution to the matrix serum to obtain a matrix serum solution added with the D-D stock solution; according to the preset concentration value of the FDP in the composite quality control product and the FDP concentration value in the matrix serum solution, adding the FDP stock solution to the matrix serum solution.

[0029] The steps of adding the D-D stock solution to the matrix serum according to the preset concentration value of the D-D in the composite quality control product and the D-D concentration value in the matrix serum in the present invention generally include: measuring the D-D concentration in the matrix serum, calculating the addition amount of the D-D stock solution according to the preset concentration value of the D-D in the composite quality control product and the measured D-D concentration value in the matrix serum; adding the D-D stock solution to the matrix serum according to the calculated addition amount of the D-D stock solution so as to adjust the D-D concentration value of different levels of the composite quality control product.

[0030] For example, assuming that the D-D concentration value in the matrix serum is a1, the preset concentration value of the D-D in the composite quality control product is a2, the D-D concentration value of the D-D stock solution is a3, and the volume of the matrix serum is V A , the addition amount or volume V of the D-D stock solution to be added B :

[0031] According to the preset concentration value of FDP in the quality control product and the FDP concentration value of the matrix serum solution, adding the FDP mother liquor to the matrix serum solution generally includes the following steps: measuring the FDP concentration of the matrix serum solution (i.e., the matrix serum added with the D-D mother liquor), calculating the addition amount of the FDP mother liquor according to the preset concentration value of FDP in the composite quality control product and the measured FDP concentration value of the matrix serum solution; adding the FDP mother liquor to the matrix serum solution according to the calculated addition amount of the FDP mother liquor, so as to adjust the FDP concentration value of the composite quality control product at different levels.

[0032] For example, assuming that the FDP concentration value of the matrix serum solution is b1, the preset concentration value of FDP in the composite quality control product is b2, the FDP concentration value of the FDP mother liquor is b3, and the volume of the matrix serum solution is V A+B , the addition amount or volume V of the D-D mother liquor to be added C。

[0033]

[0034] The preset concentration values of D-D and FDP in the composite quality control product of the present invention include the preset concentration values at the normal level and the abnormal level.

[0035] The preset concentration values of D-D and FDP at the normal level in the composite quality control product of the present invention are shown in Table 1 below: Table 1

[0036] The preset concentration values of D-D and FDP at the abnormal level in the composite quality control product of the present invention are shown in Table 2 or Table 3 below: Table 2

[0037] Table 3

[0038] In the present invention, the buffer added to human serum is composed of one or two of tris(hydroxymethyl)aminomethane, phosphate buffer, and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; the stabilizer added to human serum is composed of one or several of sucrose, trehalose, bovine serum albumin, mannitol, and sorbitol; the preservative added to human serum is composed of one or several of sodium azide, potassium azide, thimerosal, Proclin 300, and Krovin 500.

[0039] More specifically, the buffer added to human serum is composed of one or two of tris(hydroxymethyl)aminomethane at 0.01 - 1.00 mol / L, phosphate buffer at 0.01 - 1.00 mol / L, and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid at 0.5 - 30 g / L; The stabilizer added to human serum is composed of one or more of sucrose at 20 - 80 g / L, trehalose at 20 - 80 g / L, bovine serum albumin at 10 - 50 g / L, mannitol at 2 - 50 g / L, and sorbitol at 2 - 50 g / L.

[0040] The preservative added to human serum is composed of one or more of sodium azide at 0.5 - 3 g / L, potassium azide at 0.5 - 3 g / L, thimerosal at 0.5 - 3 g / L, Proclin 300 at 0.5‰ - 3‰, and Krovin 500 at 0.5‰ - 3‰.

[0041] On the other hand, the present invention also provides a blood matrix liquid D - dimer and FDP quality control product prepared by the above method.

[0042] According to the preparation method of the above composite quality control product of the present invention, the present invention first prepares a D - D mother liquor and an FDP mother liquor, and then uses the D - D mother liquor and the FDP mother liquor to prepare a composite quality control product. The following specifically illustrates the preparation process of the composite quality control product of the present invention through an example: (1) Preparation of D - D mother liquor ① Take human mixed plasma, add thrombin at 1 - 20 U / mL, and stir in a 37°C water bath for 10 - 30 min to completely coagulate the plasma, obtaining cross - linked fibrin clots; ② Take out the clots and blot dry the residual serum, and wash them repeatedly 2 - 3 times with a buffer solution with a pH of 6.5 - 8.5. The buffer solution includes but is not limited to PBS buffer solution, Tris buffer solution, and HEPES buffer solution; ③ Place the washed fibrin clots in a small amount of buffer solution, add thrombolytic drugs at 100 - 2000 U / mL, and stir in a 37°C water bath for 2 - 4 hours. The thrombolytic drugs include but are not limited to urokinase, streptokinase, staphylokinase, alteplase, reteplase, pamiteplase, tenecteplase, etc.; ④ After the fibrin clots are dissolved, sample and measure the D - D and FDP concentrations every 30 min. After the D - D concentration value is stable, that is, when the fibrin is completely degraded, inactivate it in a 56°C water bath; ⑤ Filter the inactivated solution through a 0.22 μm filter membrane and then perform ultrafiltration again to remove impurities and unstable components, collect the filtrate, mix it evenly, and measure the final D - D and FDP concentrations.

[0043] (2) Preparation of FDP mother liquor ① Take human mixed plasma, add snake venom - like thrombin at 1 - 20 U / mL, and stir in a 37°C water bath for 10 - 30 min to fully activate the coagulation system, obtaining non - cross - linked fibrin polymers; ②Centrifuge the polymer and wash it with a buffer solution with a pH of 6.5 - 8.5, repeating 2 - 3 times. The buffer solution includes but is not limited to PBS buffer solution, Tris buffer solution, and HEPES buffer solution; ③Place the washed precipitate in a small amount of buffer solution, add thrombolytic drugs at a concentration of 100 - 2000 U / mL, and stir gently in a water bath at 37°C for 2 - 4 hours. The thrombolytic drugs include but are not limited to urokinase, streptokinase, staphylokinase, alteplase, reteplase, pamiteplase, tenecteplase, etc.; ④After the precipitate dissolves, take samples every 30 minutes to measure the FDP concentration. After the FDP concentration value stabilizes, that is, when fibrin is completely degraded, add an enzyme inhibitor to inactivate the thrombolytic drug, including but not limited to one or more of aprotinin, plasminogen activator inhibitor - 1, α 2 -antifibrinolysin, aminomethylbenzoic acid, tranexamic acid, and aminoacetic acid; ⑤Filter the obtained solution through a 0.22 μm filter membrane and then perform ultrafiltration again to remove impurities and unstable components, collect the filtrate, mix it evenly, and measure the final FDP concentration.

[0044] (3) Preparation of composite quality control products ①Filter human serum through a 0.45 μm filter membrane; ②Add a buffer, a stabilizer, and a preservative to it to obtain matrix serum. The buffer is composed of one or two of 0.01 - 1.00 mol / L of tris(hydroxymethyl)aminomethane, 0.01 - 1.00 mol / L of phosphate buffer, and 0.5 - 30 g / L of 4 - (2 - hydroxyethyl)piperazine - 1 - ethanesulfonic acid; the stabilizer is composed of one or more of 20 - 80 g / L of sucrose, 20 - 80 g / L of trehalose, 10 - 50 g / L of bovine serum albumin, 2 - 50 g / L of mannitol, and 2 - 50 g / L of sorbitol; the preservative is composed of one or more of 0.5 - 3 g / L of sodium azide, 0.5 - 3 g / L of potassium azide, 0.5 - 3 g / L of thimerosal, 0.5‰ - 3‰ of Proclin 300, and 0.5‰ - 3‰ of Krovin 500;

[0045] ③Measure the D - D concentration of the matrix serum; ④Adjustment of D - D concentration: Calculate the addition amount of the D - D mother liquor according to the measured value of the matrix serum and the preset D - D concentration of the quality control product and add it, so as to adjust the D - D of different levels of quality control products to fall within the preset concentration value range shown in Table 1 or 2 or 3 below; ⑤Measure the FDP concentration of the solution obtained in step ④; ⑥Adjustment of FDP concentration: Calculate the addition amount of the FDP mother liquor according to the measured value of the solution obtained in step ④ and the preset FDP concentration of the quality control product and add it, so as to adjust the FDP of different levels of quality control products to fall within the preset concentration value range shown in Table 1 or 2 or 3 below; ⑦ The solution obtained in step ⑥ is filtered through a 0.22 μm filter membrane, mixed, and stored stably at -20°C or below after aliquoting.

[0046] Table 1

[0047] or Table 2

[0048] or Table 3

[0049] Example 1: Preparation of normal level quality control product

[0050] (1) Preparation of DD mother solution: 30 mL of human mixed plasma was added with 10 U / mL thrombin, 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 dried, and the clot was washed three times with a PBS buffer solution of pH 7.4; the washed fibrin clot was placed in 5 mL of buffer solution, 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 which the DD and FDP concentrations were measured every 30 min. After the DD concentration value was basically stable, i.e., 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: Table 4

[0051] (2) Preparation of FDP mother solution: 30 mL of human mixed plasma was added with 10 U / mL snake venom thrombin, and stirred in a 37°C water bath for 30 min to obtain non-cross-linked fibrin polymers; the polymers were centrifuged and washed with a pH 7.4 PBS buffer, and this was 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 h. During this period, samples were taken every 30 min to measure the FDP concentration. When the FDP concentration value stabilized, i.e., when the fibrin was completely degraded, α 2 - Anti-plasmin (5U / mL) and plasminogen activator inhibitor-1 (100U / mL) inactivated activated plasmin and added urokinase, stirred in a 37°C water bath for 30 min, filtered the solution with a 0.22μm filter membrane and then ultrafiltered again, collected the filtrate, mixed it, and determined the final FDP concentration. The specific results are shown in Table 5: Table 5

[0052] (3) Preparation of DD and FDP composite quality control products ① Take 1 L of human serum and filter it through a 0.45 μm filter membrane to obtain filtered serum; ② Add buffer, stabilizer, and preservative to the filtered serum according to Table 6 to obtain matrix serum: Table 6

[0053] ③ Measure the D-D concentration of the matrix serum to be 0.24 mg / L; ④ Calculate and add 53 μL of D-D mother liquor according to the measured value of the matrix serum and the preset D-D concentration (0.4 mg / L) of the quality control product; ⑤ Measure the FDP concentration of the solution obtained in step ④ to be 1.2 mg / L; ⑥ Calculate and add 434 μL of FDP mother liquor according to the measured value of the solution obtained in step ④ and the preset FDP concentration (3.5 mg / L) of the quality control product; ⑦ Filter the solution obtained in step ⑥ through a 0.22 μm filter membrane and mix it to obtain a normal-level quality control product; ⑧ Detect that the final concentrations of D-D and FDP in the quality control product are 0.38 mg / L and 3.6 mg / L respectively; ⑨ Mix well, aliquot, and store stably at -20 °C or below.

[0054] Example 2: Preparation of an abnormal-level quality control product The difference from Example 1 is that the preset D-D concentration is 1.5 mg / L and the preset FDP concentration is 10 mg / L. Accordingly, the amounts of D-D mother liquor and FDP mother liquor added are calculated to be 420 μL and 1245 μL respectively, and the D-D and FDP concentrations of the obtained quality control product are 1.46 mg / L and 9.7 mg / L respectively, with other conditions being the same.

[0055] Comparative Example 1: Preparation of a normal-level quality control product The difference from Example 1 is that the preparation volume is 10 mL, and only the self-made D-D mother liquor or the purchased FDP raw material (FDP concentration is 1.21 mg / mL) is added. When only the self-made D-D mother liquor is added, when the D-D concentration is within the normal reference range, the FDP concentration is lower than the lower limit of the linear range of the kit, and the precision and quality control effect of the quality control product cannot be guaranteed. When the addition amount is increased to make the FDP concentration meet the requirements, the D-D concentration has exceeded the normal reference range; when only the purchased FDP raw material is added, the same situation exists, indicating that this raw material contains D-D components. The specific results are shown in Table 7: Table 7

[0056] The results show that Examples 1 and 2 can achieve DD and FDP concentrations that meet the requirements of normal or abnormal quality control levels at the same time; while Comparative Example 1 cannot prepare a normal level quality control product with DD and FDP concentrations within the normal reference range.

[0057] Uniformity test of quality control products in Example 1 and Example 2 The examples 1 and 2 of the present invention and the commercially available quality control products (normal DD quality control products, abnormal DD quality control products, normal FDP quality control products, abnormal FDP quality control products, all of which are freeze-dried dosage forms with buffer matrix) are re-melted or accurately re-dissolved in the indicated volume of distilled water, and the uniformity test is performed according to the following method: 10 minimum packaging units of quality control products are randomly selected for each quality control product and randomly numbered 1 to 10, and each packaging unit is measured 3 times. Considering the random variation of the measurement system caused by factors such as time, the 3 measurements are 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 …………………Formula (1) …………………………Formula (2) …………………Formula (3) ………………………………………… Formula (4) …………………………………………Formula (5) ……………Formula (6) ……………………Formula (7) ……………Formula (8) ………………………………Formula (9) …………………………Formula (10) …………………………………………Formula (11) Where: SS 总和 、 SS 瓶间 、 SS 瓶内 — are the total variance, between-bottle variance, and within-bottle variance respectively; MS 瓶间 、 MS 瓶内 — between-bottle mean square and within-bottle mean square; x i — the i-th measured value or calculated result of the specified parameter; — the total average; n i — the number of repeated determinations of sample i; x ij — the j-th result of sample i; — degrees of freedom; F — F test value; n 0 — the number of valid measurements; α — the number of samples drawn; N — the total number of tests; S bb — between-bottle standard deviation; S r — within-bottle standard deviation (repeatability standard deviation).

[0058] Table 8

[0059] By calculating the test data and referring to the table, it can be seen that , , , F 0.05(v1,v2) = 2.3928, and the F values of all quality control products D-D and FDP are all < F 0.05(v1,v2) , indicating good between-bottle uniformity. And the CV 瓶间 of D-D and FDP in Example 1 and Example 2 are all < the CV 瓶间 of the corresponding items of commercially available quality control products, proving that the repeatability and precision of the quality control products of the present invention are superior to those of commercially available quality control products.

[0060] 6.6 Stability Test of the Quality Control Products in Example 1 and Example 2 after Opening the Vial The quality control products of Example 1 and Example 2 of the present invention and commercially available quality control products (normal D-D quality control product, abnormal D-D quality control product, normal FDP quality control product, abnormal FDP quality control product, all freeze-dried dosage forms with buffer matrix) were thawed 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 D-D and FDP. According to t the inspection table, a significance test for the slope trend was carried out. For a 95% confidence level, when ∣ b 1 ∣ < t 0.05,n-2 × s( b 1 ) it indicates that the change trend of the stability result of the quality control product is not significant; otherwise, the change trend of the stability result is significant. The test results are shown in Tables 9 - 10: Table 9 t Inspection Table

[0061] Table 10

[0062] The detection data was analyzed for trend test according to the t-test table. The results showed that after thawing, the D-D and FDP combined quality control products prepared by the present invention were stored at 2°C - 8°C for 9 days, and there was no significant change trend for both D-D and FDP. However, for the normal value and abnormal value quality control products from commercial sources, after reconstitution and evaluation under the same conditions, both D-D and FDP showed a trend of decreasing. This fully proves that the quality control products of the present invention have better stability than commercial quality control products.

[0063] Although the present invention has been described in detail above, the present invention is not limited thereto. Those skilled in the art of this technology can make various modifications according to the principle of the present invention. Therefore, all modifications made according to the principle of the present invention should be understood to fall within the protection scope 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 following steps: adding thrombin to human mixed plasma to obtain a cross-linked fibrin clot, and subjecting the obtained cross-linked fibrin clot to a treatment including washing, dissolving, inactivating and ultrafiltration to obtain a DD mother solution; The non-cross-linked fibrin polymer is obtained by adding snake venom thrombin to human mixed plasma, and the obtained non-cross-linked fibrin clot is 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 products are obtained by respectively adding the DD mother solution and the FDP mother solution to the matrix serum, filtering and freezing the matrix serum added with the DD mother solution and the FDP mother solution.

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, adding the DD mother solution to the matrix serum according to the preset concentration value of the quality control product DD and the matrix serum DD concentration value, comprising: Determine the matrix serum DD concentration, and calculate the amount of the DD mother solution to be added according to the preset concentration values ​​of DD and FDP composite quality control product DD and the determined matrix serum DD concentration value; The DD mother solution is added to the matrix serum according to the calculated addition amount of the DD mother solution, so as to adjust the DD concentration values ​​of the composite quality control products of different levels of DD and FDP.

4. The preparation method according to claim 2, adding the FDP mother solution to the matrix serum solution according to the preset concentration value of the quality control product 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 mother solution to be added according to 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 added, the FDP stock solution is added to the matrix serum solution to adjust the FDP concentration values ​​of the composite quality control products of 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 mixed human 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 the cross-linked fibrin clot obtained comprise: The residual serum from the cross-linked fibrin clot was aspirated and washed repeatedly with a buffer having a pH of 6.5-8.5; The washed cross-linked fibrin clot is placed in a buffer solution, a thrombolytic drug is 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 clots are dissolved, samples are taken at regular intervals to determine 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; 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 step of adding snake venom thrombin to human mixed plasma to obtain non-crosslinked fibrin polymer comprises: 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 clots including washing, precipitation dissolution, inactivation and ultrafiltration comprises: After the non-crosslinked fibrin polymers were centrifuged to obtain a precipitate, they were repeatedly washed with a buffer solution at 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, and after the FDP concentration value is stabilized, i.e., after 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(hydroxymethyl)aminomethane, 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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