D-dimer and fdp complex quality control product and preparation method and application thereof

Abstract

This invention discloses a D-dimer and FDP composite quality control product, its preparation method, and its application. The preparation method includes the following steps: S1, mixing plasma with a coagulation activator to coagulate the plasma; S2, enzymatically hydrolyzing the coagulated plasma to obtain a D-dimer and FDP composite mother liquor; wherein the enzymes used for enzymatic hydrolysis include tissue plasminogen activator, glucosinolate, and heparin; S3, diluting the D-dimer and FDP composite mother liquor with a protective solution to obtain the D-dimer and FDP composite quality control product. The composite quality control product prepared by this invention can be used to prepare in vitro diagnostic reagents. In clinical practice, it can be used not only for testing D-dimer or FDP individually, but also for their combined detection, saving costs, shortening detection time, and improving detection efficiency.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a D-dimer and FDP composite quality control product, its preparation method, and its application. Background Technology

[0002] D-dimer and fibrin degradation products (FDPs) are degradation products of fibrin or fibrinogen in the human body after fibrin(ogen) dissolution, produced by plasminogen activating the coagulation system and fibrin(ogen) dissolution. When the coagulation system is activated, fibrinogen is cleaved to form fibrin monomers. These monomers are then converted into cross-linked fibrin under the catalysis of activated coagulation factor XIII. Plasminogen is converted into plasmin under the action of plasminogen activator. Plasmin dissolves fibrin(ogen), producing various soluble fragments, forming FDPs. D-dimer is a component of FDPs. It is the final product containing two D fragments linked by a Y chain, produced by the cross-linking of fibrin monomers with activated coagulation factor XIII to form stable cross-linked fibrin, followed by hydrolysis by plasmin. It is the preferred molecular marker reflecting hypercoagulability and fibrinolytic activation in the body.

[0003] In recent years, D-dimer has become a routine clinical coagulation test. Due to its high sensitivity and predictive ability, it is of significant value in the differential diagnosis and progression of many diseases, such as venous thrombosis, disseminated intravascular coagulation (DIC), preeclampsia, and malignant tumors, as well as in monitoring thrombolytic therapy and prognosis. When thrombosis occurs, the fibrinolytic system is also activated. At this time, plasmin can degrade fibrinogen and fibrin monomers to form smaller fragments, namely FDP. Therefore, FDP is both a screening indicator of fibrinolytic activity and a detection indicator for secondary fibrinolysis in the coagulation system. It is often used in conjunction with D-dimer in clinical applications for diseases related to the imbalance between coagulation and fibrinolysis. For example, it plays an important role in differentiating between primary and secondary hyperfibrinolysis, DIC, malignant tumors, cardiovascular diseases, liver diseases, preeclampsia, and preeclampsia, as well as in monitoring disease severity, progression, treatment, and prognosis.

[0004] Although D-dimer and FDP testing are widely used clinically, the diversity of testing methods makes comparability between different methods poor. Furthermore, there are currently no unified international or national standards, and the determination of quality levels during continuous dynamic testing has not yet been standardized, affecting the accuracy of clinical test results. Clinical laboratories in medical institutions should conduct internal quality control for the tests they perform. The purpose is to monitor the testing process, evaluate the reliability of test results, and determine whether test reports can be issued normally; this is a necessary measure to ensure high-quality operation. In the process of internal quality control, the quality of control materials is crucial. Currently, D-dimer and FDP control materials are manufactured by various companies, but the quality varies, with differences between different batches from the same manufacturer, and materials from different manufacturers are not interchangeable. Due to the lack of international and national standards, quality monitoring of testing items in clinical laboratories faces many problems. Therefore, establishing internal quality control materials in laboratories will help us monitor the quality of D-dimer and FDP test results in routine clinical trials.

[0005] Patent CN201410619619.0 discloses a method for preparing a D-dimer and FDP composite quality control product, comprising the following steps: adding thrombin to plasma in the presence of calcium ions to completely coagulate the plasma; subsequently, crushing the coagulated plasma, adding an enzyme solution (containing plasminogen and glucosinolate) to degrade fibrin; terminating the enzymatic reaction after the reaction is complete; diluting the obtained D-dimer and FDP composite mother liquor with a protective solution to obtain the D-dimer and FDP composite quality control product; the difference between each quality control product in batch-to-batch variation testing is within 10%; in stability testing, the difference between groups remains within 5% within 7 days after opening; and it can be stored sealed at 2-8℃ for 24 months. The inter-group differences remained within 10%, meeting the quality control requirements. Patent CN201710861687.1 also discloses a method for preparing a D-dimer and FDP composite quality control product. Fresh blood is poured into a glass container containing glass beads to obtain white fibrinogen. After reconstitution, it is enzymatically hydrolyzed with protease to obtain a D-dimer and FDP composite mother liquor. It is then diluted with a protective solution to obtain the D-dimer and FDP composite quality control product. The differences between the quality control products tested for batch-to-batch variation were within 5%. In the stability test, the inter-group differences remained within 5% within 7 days after opening the bottle, and the inter-group differences also remained within 5% within 14 months of sealed storage at 2-8℃, meeting the quality control requirements. However, in the above methods for preparing D-dimer and FDP composite quality control products, the addition of thrombin to promote coagulation leads to the formation of similar isomers due to differences in animal origin and prothrombin cleavage sites. This results in excessively slow clotting rates and uneven cross-linking during fibrin formation. Furthermore, D-dimer and FDP composite quality control products are generally stored at 2-8°C or under low-temperature freezing conditions. The active ingredients are unstable at room temperature, resulting in a very short product shelf life (less than 10 hours), causing significant inconvenience to product storage and transportation. Therefore, this invention is proposed. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention discloses a method for preparing a composite quality control product of D-dimer and FDP, which avoids the formation of isomers that affect the test results, ensures stable product quality, and exhibits excellent stability at low temperatures, room temperatures, and even high temperatures, with inter-group differences remaining within 5%.

[0007] The first objective of this invention is to provide a method for preparing a D-dimer and FDP composite quality control sample, comprising the following steps:

[0008] S1. Mix plasma with a coagulation activator to coagulate the plasma;

[0009] S2. Enzymatically hydrolyze the coagulated plasma to obtain a complex mother liquor of D-dimer and FDP; wherein the enzymes used in the enzymatic hydrolysis include tissue plasminogen activator (t-PA), succinyl kinase (SAK), and heparin;

[0010] S3. Dilute the D-dimer and FDP composite mother liquor with a protective solution to obtain the D-dimer and FDP composite quality control product.

[0011] Furthermore, the coagulation activator includes calcium ions, phospholipids, and tissue factors.

[0012] Furthermore, phospholipids include, but are not limited to, natural phospholipids and synthetic phospholipids.

[0013] Furthermore, natural phospholipids can be selected from soybean phospholipids or rabbit brain phospholipids.

[0014] Furthermore, phospholipids are synthesized by at least two of the following substances: acylserine, phosphatidylethanolamine, phosphatidylcholine, phosphatidylglycerol, and cholesterol.

[0015] Furthermore, the coagulation activator consists of: calcium ions, not limited to calcium chloride 0.5-5% w / v, calcium lactate 0.5-5%; phospholipids, not limited to soybean phospholipids 0.1 mg / L-50 mg / L, rabbit brain phospholipids 0.5 mg / L-100 mg / L, synthetic phospholipids 0.2 mg / L-20 mg / L; and activators, not limited to rabbit brain tissue factor 10 ug / L-200 ug / L, embryonic tissue factor 5 ug / L-80 ug / L, and synthetic tissue factor 2 ug / L-60 ug / L.

[0016] Furthermore, the enzymatic hydrolysis system contains 10-100 ug / L tissue plasminogen activator (t-PA), 20-200 ug / L glucokine, and 500-1500 U / L heparin.

[0017] Furthermore, the weight-average molecular weight of heparin is in the range of 1.2kD-40kD.

[0018] Furthermore, the protective solution comprises: buffer solution, stabilizer, preservative, and surfactant.

[0019] Furthermore, the composition of the protective fluid is as follows:

[0020] Buffer solutions: 10-100mM Tris-HCl buffer, 0.2-10g / L imidazole-HCl buffer, 10-100mM Hepes Na-HCl buffer, 10-100mM phosphate buffer, or 5-25g / L 4-hydroxyethylpiperazine ethanesulfonic acid;

[0021] Stabilizers: 1%-10% sucrose, 1%-10% trehalose, 2%-8% sorbitol, 1%-5% mannitol, 1%-5% glycine, 0.5%-6% polyethylene glycol, 1%-3% dextran, 0.3%-5% bovine serum albumin or 0.5%-1.5% sodium chloride;

[0022] Surfactant: 0.02%-0.5% TWEEN 80 / 60 / 20, or 0.05%-0.4% Triton-X100;

[0023] Preservatives: 0.5-5 g / L NaN3 or 1-5 mL / L PC-300 / 950.

[0024] Further, in step S1, the plasma and coagulation activator are incubated at 30-40°C (preferably 37°C) to coagulate the plasma.

[0025] Further, in step S1, the volume ratio of plasma to coagulation activator is 0.5-1:1-5.

[0026] Furthermore, in step S2, after the enzymatic hydrolysis is completed, the mixture is heated at 50-100℃ for 0.5-10h to terminate the enzymatic hydrolysis reaction.

[0027] Further, the D-dimer and FDP composite mother liquor obtained in step S2 is diluted with a protective solution at a dilution factor of 100-500 times.

[0028] The second objective of this invention is to provide a quality control product of D-dimer and FDP composite prepared by the above preparation method.

[0029] A third objective of this invention is to provide the application of the above-mentioned D-dimer and FDP composite quality control material in the preparation of coagulation test reagents.

[0030] Furthermore, the coagulation test reagent may be a D-Dimer test reagent.

[0031] Furthermore, the coagulation test reagent may be a fibrin degradation product (FDP) test reagent.

[0032] By means of the above-described solution, the present invention has at least the following advantages:

[0033] This invention prepares D-dimer and FDP quality control samples by activating the coagulation and fibrinolysis systems, and further measures the homogeneity and stability of the quality control samples. The results show that the D-dimer and FDP composite quality control samples exhibit good homogeneity and stability, making them suitable for internal quality control in clinical laboratories. Furthermore, the raw materials selected in this invention are readily available, the preparation process is simple and inexpensive, and it can be used simultaneously for D-dimer and FDP detection. The homogeneity and stability of the quality control samples meet the requirements, facilitating internal quality control, saving costs, and improving testing quality.

[0034] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the preferred embodiments of the present invention are described below. Detailed Implementation

[0035] The present invention will be further described below with reference to specific embodiments, so that those skilled in the art can better understand and implement the present invention, but the embodiments are not intended to limit the present invention.

[0036] Example 1: Preparation of quality control samples

[0037] (1) Preparation of plasma samples

[0038] Blood samples anticoagulated with 109 mmol / L sodium citrate at a 9:1 ratio, D-dimer positive samples, and normal human samples were all obtained from Kowloon Hospital affiliated with Shanghai Jiao Tong University School of Medicine. Hepatitis B, hepatitis C, syphilis, and human defective viruses were all negative, and the samples showed no lipemia or hemolysis. After centrifugation at 3000g for 15 minutes, platelet-poor plasma (platelet count ≤10*10^6) was obtained. 9 / L).

[0039] (2) Preparation of activator solution

[0040] Preparation of human thrombin activator A: Mix 2 g / L calcium chloride and 10000 U / L human thrombin;

[0041] Preparation of porcine thrombin activator B: Mix 2 g / L calcium chloride and 10000 U / L porcine thrombin;

[0042] Preparation of bovine thrombin activator C: Mix 2 g / L calcium chloride and 10000 U / L bovine thrombin;

[0043] Preparation of tissue factor activator D: Mix 2 g / L calcium chloride, 10 mM rabbit brain phospholipids, and 20 ug / L rabbit brain tissue factor.

[0044] (3) Preparation of enzyme solution

[0045] Enzyme solution A: Mix 20 μg / L plasmin and 40 μg / L styrokinase (SAK).

[0046] Enzyme solution B: Mix 20 μg / L plasminogen and 40 μg / L styrokinase (SAK);

[0047] Enzyme solution C: Mix 20 μg / L tissue plasminogen activator (t-PA) and 40 μg / L succinyl kinase (SAK);

[0048] Enzyme solution D: Mix 20 μg / L tissue plasminogen activator (t-PA), 40 μg / L styraxokinase (SAK), and 1000 U / L heparin (molecular weight 15 kD).

[0049] (4) Preparation of diluent

[0050] Prepare a dilution solution using 25 mmol / L, pH 7.4 PBS, 60 g / L sucrose, 20 g / L polyethylene glycol 8000, 10 g / L LBSA, and 0.5 g / L NaN3.

[0051] (5) Preparation of D-dimer and FDP composite mother liquor

[0052] S1. Add the activator to the plasma obtained in step (1), mix by inverting, and bathe in a water bath at 37°C for half an hour to coagulate the plasma.

[0053] S2. Add enzyme solution to coagulated plasma, shake to mix, and place in a shaker at 37°C for 4-8 hours. When removed, fibrin has been degraded. Finally, place the plasma in a 56°C water bath for 45 minutes to inactivate coagulation factors, anticoagulants, and fibrinolytic enzymes and proteins, to obtain D-dimer and FDP complex mother liquor.

[0054] (6) Preparation of D-dimer and FDP composite quality control material

[0055] The concentration of the D-dimer and FDP composite mother liquor was adjusted using a diluent until the D-dimer content reached 4.0 mg / L, thus obtaining the D-dimer and FDP composite quality control product, which was then aliquoted and sealed for storage in a 4°C refrigerator for later use.

[0056] Example 2: Potency results of D-dimer and FDP composite quality control samples prepared under different conditions

[0057] (1) Results of preparing D-dimer and FDP composite quality control samples with four different activators

[0058] Under the same plasma samples and conditions (enzyme solution D was used in the enzymatic digestion process), D-dimer and FDP composite quality control samples were prepared using four different activators. To match the detection range of the reagents, the prepared quality control samples were diluted. The quality control sample prepared with human thrombin was diluted 200 times, the quality control sample prepared with porcine thrombin was diluted 100 times, the quality control sample prepared with bovine thrombin was diluted 50 times, and the quality control sample prepared with tissue factor activator was diluted 300 times. The results are shown in Table 1. It shows that the D-dimer and FDP composite quality control sample prepared with tissue factor activator (activator D) has the highest potency and the closest proportion to the abnormal plasma samples.

[0059] Table 1. Preparation of D-dimer and FDP composite quality control samples using four different activators.

[0060]

[0061] (2) Results of preparing D-dimer and FDP composite quality control samples by lysing fibrin clots with four enzyme solutions

[0062] In the fibrin clot prepared with tissue factor activator, D-dimer and FDP composite quality control products obtained using four different lytic enzymes are shown in Table 2. The results show that the lysis effect of the mixed lysis solution of tissue-type plasminogen activator and staphylokinase is the best and the titer is the highest.

[0063] Table 2 Preparation of D-dimer and FDP composite quality control products with four enzyme solutions

[0064]

[0065] Example 3 Homogeneity and stability tests

[0066] (1) Performance evaluation method

[0067] A. Homogeneity evaluation: The evaluation is carried out according to the homogeneity test "4.2 One-way ANOVA" in CNAS-GL03. Randomly select 10 quality control products and randomly number them 1-10. Each is tested 3 times on the detection system. Considering the random variation caused by factors such as time in the measurement system, the 3 measurements are carried out in different orders, such as 1, 5, 7, 10, 2, 3, 6, 7, 8, 9. Calculate the F value according to the results. If the calculated F value < F critical value (α = 0.05), it indicates that there is no statistical difference within and between samples, and the samples are homogeneous.

[0068] B. Stability test: Place the quality control products at -20°C and 4°C, and detect them once a month for 25 consecutive months. The average value of each 3 detections is defined as the measured value, and calculate CV%; place the quality control products at 37°C, detect them once a day for 7 consecutive days, and the average value of each 3 detections is defined as the test value, and calculate CV%.

[0069] C. Statistical processing: Use SPSS 23.0 software for statistical processing. One-way ANOVA is used for homogeneity evaluation, and t-test is used for stability evaluation. There is statistical significance when p ≤ 0.05.

[0070] (2) Performance test results of D-dimer and FDP composite quality control products

[0071] (The test samples are four D-dimer and FDP composite quality control products prepared with activator D and enzyme solution D in Example 2)

[0072] A. Homogeneity evaluation of quality control products

[0073] Calculate the statistical quantity F of the quality control product, and check the F table to obtain the critical value F 0.05 (2, 8) = 4.46, F < F 0.05 (2, 8) = 4.46, there is no statistical difference in uniformity between bottles.

[0074] Table 4 Analysis of Uniformity Results

[0075]

[0076] Note: SS 总 Variance; MS: Mean Square

[0077] B. Stability test results of quality control products

[0078] ①Stability test results at -20℃: As shown in Table 5, the coefficient of variation of the quality control sample was less than 5% for each test at -20℃, which is within the acceptable range; and after being stored at -20℃ for 25 months, there was no significant difference in the results (P>0.05).

[0079] Table 5. Test results of quality control samples after being stored at -20℃ for 24 months.

[0080]

[0081]

[0082] Note: The numbers in the table are the average of three measurements.

[0083] ②Stability test results at 4℃: As shown in Table 6, the coefficient of variation of the quality control sample was less than 5% for each test at 4℃, which is within the acceptable range; and after 25 months of storage at 4℃, there was no significant difference in the results (P>0.05).

[0084] Table 6. Test results of quality control samples after 25 months of storage at 4℃.

[0085]

[0086]

[0087] Note: The numbers in the table are the average of three measurements.

[0088] ③Stability test results at 37℃: As shown in Table 7, at 37℃, the coefficient of variation of the quality control sample was less than 5% for each test, which is within the acceptable range; and after 7 days at 37℃, there was no significant difference in the results (P>0.05).

[0089] Table 7. Test results of quality control samples after being placed at 37℃ for 7 days.

[0090]

[0091] Note: The numbers in the table are the average of three measurements.

[0092] Stability results show that the D-dimer and FDP quality control products prepared in this experiment have good stability and meet the requirements of internal quality control products.

[0093] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for preparing a D-dimer and FDP complex quality control, characterized by, Includes the following steps: S1. Mixing plasma with a coagulation activator to coagulate the plasma; the coagulation activator is a composition of calcium ions, phospholipids, and tissue factors; S2. Enzymatically hydrolyze coagulated plasma using an enzyme solution to obtain a D-dimer and FDP complex mother liquor; wherein the enzyme solution contains 10-100 ug / L tissue plasminogen activator, 20-200 ug / L glucokine and 500-1500 U / L heparin with a molecular weight of 15kD; S3. Dilute the D-dimer and FDP composite mother liquor with a protective solution to obtain the D-dimer and FDP composite quality control product.

2. The method of claim 1, wherein: The protective solution includes buffers, stabilizers, preservatives, and surfactants.

3. The method of claim 2, wherein: The stabilizer is selected from one or more of sucrose, trehalose, sorbitol, mannitol, glycine, polyethylene glycol, dextran, bovine serum albumin, and sodium chloride.

4. The method of claim 1, wherein: In step S1, the volume ratio of plasma to coagulation activator is 0.5-1:1-5.

5. The D-dimer and FDP composite quality control product prepared by the preparation method according to any one of claims 1-4.

6. The application of the D-dimer and FDP composite quality control material as described in claim 5 in the preparation of coagulation test reagents.

7. Use according to claim 6, characterized in that: The coagulation test reagent is a D-dimer test reagent.

8. Use according to claim 6, characterized in that: The coagulation test reagent is a fibrin degradation product test reagent.

Images