A method for preparing a D-dimer and FDP composite quality control sample

Abstract

This invention discloses a method for preparing a D-dimer and FDP composite quality control sample, belonging to the field of diagnostic reagent technology. The method includes the following steps: Step 1: Weigh the cryoprecipitate, add it to buffer solution, melt it in a 37°C water bath, and collect the supernatant by centrifugation; Step 2: Add trypsin, calcium chloride solution, and thrombin, mix well, and let it stand in a 37°C water bath; Step 3: Detect the FDP and DD content using an automated coagulation analyzer; Step 4: Terminate the reaction with a stop solution; Step 5: Prepare the composite quality control sample according to the target content and ratio of DD and FDP. This method is simple, low-cost, and yields extremely high concentrations of D-dimer and FDP. The prepared D-dimer and FDP composite quality control sample has an adjustable ratio, good stability, and broad applicability to reagents and instruments.

Description

Technical Field

[0001] This invention relates to the field of testing reagent technology, specifically to a method for preparing a composite quality control product of D-dimer and FDP. Background Technology

[0002] D-dimer and FDP quality control products are used to monitor the controlled detection of FDP and DD-dimer in coagulation analyzers.

[0003] CN104458367A and CN201710861687 have disclosed methods for preparing composite quality control products of D-dimer and FDP. The former involves coagulating fresh bovine plasma with calcium and thrombin, then crushing it, and adding plasminogen and glucosamine (using glucosamine to activate plasmin, which then degrades fibrin). The latter involves treating rabbit plasma to produce fibrinogen, removing and resolving the fibrinogen, adding protease to degrade fibrin, and finally diluting both with enzyme-terminating solutions at 50℃~100℃ to obtain composite quality control products.

[0004] However, fibrin coagulates into a colloid, making it difficult for plasmin in the liquid to penetrate the fibrin quickly even after crushing, hindering degradation. Without enzyme activity termination, FDP and D-dimers exhibit poor stability, and their ratio becomes uncontrollable. Using high temperatures to terminate enzyme activity also causes denaturation and aggregation of FDP and D-dimers, affecting their natural stable state. The processes of crushing plasma or separating fibrinogen from plasma, terminating enzyme activity with high temperatures, and adding plasmin and various kinases increase the complexity of the process, extending production time and cost. Furthermore, plasma concentration limits the concentration of intermediate FDP / DD composite quality control products. Summary of the Invention

[0005] To achieve the above effects, the present invention provides the following technical solution: a method for preparing a D-dimer and FDP composite quality control product, comprising the following steps:

[0006] Step 1: Weigh the cryoprecipitate, add it to the buffer solution, melt it in a 37°C water bath, and collect the supernatant by centrifugation.

[0007] Step 2: Add trypsin, calcium chloride solution and thrombin, mix well, and let stand in a 37°C water bath.

[0008] Step 3: The FDP and DD levels are tested using a fully automated coagulation analyzer.

[0009] Step 4: Terminate the reaction with the stop solution.

[0010] Step 5: Prepare the quality control product according to the target content and ratio of the composite quality control product DD and FDP.

[0011] Furthermore, according to the operating steps in step one, the 218 g of cryoprecipitate was added to 200 ml of buffer solution.

[0012] Furthermore, according to the operating steps in step one, the water bath at 37 degrees Celsius is used for melting for 2-4 hours, and the centrifugation is performed at 4000 rpm at 4 degrees Celsius for 10 minutes.

[0013] Further, according to the operation steps in step two, 2 ml of 0.1-10% trypsin is added to the 200 ml of cryoprecipitate buffer and mixed well, 5 ml of calcium chloride is added and mixed well, 2 ml of 0.1-1 mg / ml thrombin is added and mixed well, and then placed in a 37°C water bath and left to stand for 3-78 hours.

[0014] Furthermore, according to the operating steps in step three, the fully automated coagulation analyzer is model CX-9000.

[0015] Furthermore, according to the operation steps in step four, the termination liquid is a isolate of a naturally occurring substance.

[0016] Furthermore, according to the operating steps in step four, the terminating solution contains 2.5% preservative 1, 0.05% preservative 2, and 5% protective agent.

[0017] Furthermore, following the steps in step four, add an equal volume of stop solution to the reaction solution and react at room temperature for 5–30 minutes.

[0018] Furthermore, according to the operating steps in step five, prepare the composite quality control product according to the target content and proportion.

[0019] This invention provides a method for preparing a D-dimer and FDP composite quality control product, which has the following advantages: the preparation method of this D-dimer and FDP composite quality control product is simpler, lower in cost, uses natural raw materials with higher utilization rate, has a convenient enzyme activity termination technology, produces a higher concentration of D-dimer and FDP composite quality control product with controllable ratio, and has good stability. It can meet the needs of more coagulation testing instruments as much as possible. This invention provides a simpler, faster, more universal, and more stable intermediate. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the preparation process of the present invention. Detailed Implementation

[0021] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0022] Example 1

[0023] This embodiment provides a method for preparing quality control samples of D-dimer and FDP, including the following steps:

[0024] 1) Resolution and centrifugation of cryoprecipitate:

[0025] Weigh 21.8 g of cryoprecipitate, add 20 mL of Tris buffer, centrifuge at 4000 rpm for 10 minutes, collect the clear supernatant, and discard the precipitate.

[0026] 2) Fibrin coagulation and degradation: Take 2 ml of the reconstituted cryoprecipitate, add 20 μL of 0.8% trypsin, mix well, add 50 μL of 0.2 mmol / L calcium chloride and mix well, add 20 μL of 0.1 mg / mL thrombin and mix well, coagulate in a 37°C water bath, and continue in the water bath until dissolved for 12 hours.

[0027] 3) Hydrolysis termination: Add an equal volume of termination plasma, that is, add 2 ml of reconstitution solution to 2 ml of termination plasma and test. The results are shown in the table below.

[0028] Table 1:

[0029] Degradation time FDP / c DD / c FDP:DD 12 hours 18060 2090 8.6

[0030] 4) Add 55.6 μL of the stop solution to 50 mL of abnormal quality control plasma, take 1 mL of the solution in three replicates and test and calculate the mean. Mix well, dispense and freeze dry, take three bottles for testing and calculate the mean. The results are shown in the table below.

[0031] Table 2:

[0032]

[0033] Example 2: The effect of fibrin content on FDP and DD.

[0034] The preparation method is the same as in Example 1, except that: 2 mL of cold precipitation reconstitution solutions of 0.54 g / mL, 1.09 g / mL, and 1.64 g / mL were prepared respectively, 20 μL of 8% trypsin was added, mixed well, 50 μL of 0.2 mmol / L calcium chloride was added and mixed well, 20 μL of 0.1 mg / mL thrombin was added and mixed well, and the mixture was solidified in a 37°C water bath for 12 hours until dissolved.

[0035] Table 3:

[0036]

[0037] The table shows that the concentration of cryoprecipitate has little effect on the FDP:DD ratio after degradation, but the concentration itself has a significant impact. However, excessively high concentrations slow down the degradation rate and prolong the reaction time. Therefore, adding 1.09 g / mL cryoprecipitate to 20 μL of 0.8% trypsin, mixing well, then adding 50 μL of 0.2 mmol / L calcium chloride and mixing well, and finally adding 20 μL of 0.1 mg / mL thrombin and mixing in a water bath, yields a higher FDP yield.

[0038] Example 3: Effect of trypsin concentration on FDP and DD.

[0039] Table 4:

[0040]

[0041] The preparation method is the same as in Example 1, except that: 2 ml of cold precipitation reconstitution solution was used to prepare 0.2%, 0.4%, 0.8%, and 1% trypsin, respectively. 20 μL of trypsin of different concentrations were mixed, 50 μL of 0.2 mmol / L calcium chloride was added and mixed, 20 μL of 0.1 mg / mL thrombin was added and mixed, and the mixture was solidified in a 37°C water bath and continued in the water bath until dissolved for 12 hours.

[0042] The results above show that 0.8% can be degraded within 12 hours and reaches a certain concentration.

[0043] Example 4: Effect of degradation time on FDP and DD

[0044] Table 5:

[0045]

[0046] Example 5: The impact of termination methods on FDP and DD

[0047] Take 2 mL of the cryoprecipitate reconstituted into three small glass vials. Add 20 μL of 10% trypsin (containing 0.46% EDTA) to each vial and mix well. Then add 100 μL of 0.2 mol / L calcium ions and mix well. Finally, add 20 μL of 0.3 mg / mL thrombin, quickly cap the vials, invert and mix well, then incubate in a 37°C water bath.

[0048] After 28 hours of hydrolysis, one bottle of hydrolysate was removed. From one bottle, 1 ml of hydrolysate was added to 1 ml of terminated plasma and mixed thoroughly. 1 ml of the terminated plasma solution was stored at -20°C as a control. The remaining terminated plasma solution was kept in a water bath. From the other bottle, 0.5 ml of hydrolysate was added to 2.5 ml of terminated plasma. 1 ml of the terminated plasma solution was stored at -20°C as a 28-hour control. The remaining terminated plasma solution was kept in a water bath. The remaining hydrolysate was stored at -20°C as a 28-hour control.

[0049] The remaining two bottles of hydrolysate were kept in a water bath for 48 hours. All solutions were then removed and diluted 10,000 times for testing.

[0050] Table 6:

[0051]

[0052] The results show that adding an equal volume of stop plasma can inactivate trypsin.

[0053] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a D-dimer and FDP complex quality control, characterized by, The method comprises the following steps: S1, weigh the cold precipitate and add buffer, melt in a 37-degree Celsius water bath, and collect the supernatant by centrifugation; S2, add 20 microliters of 0.8% trypsin, 50 microliters of 0.2 millimoles / liter calcium chloride, and 20 microliters of 0.1 milligrams / milliliter thrombin, mix well, and stand in a 37-degree Celsius water bath; S3, detect the FDP and DD content by using a full-automatic coagulation instrument; S4, terminate the reaction by using a termination solution; S5, prepare the quality control product according to the target content and proportion of the DD and FDP quality control product.

2. The method of claim 1, wherein the D-dimer and FDP calibrator is prepared by the steps of: The method comprises the following steps: According to the operation steps in S1, 218 grams of the cold precipitate are added into 200 milliliters of buffer.

3. The method of claim 1, wherein the D-dimer and FDP calibrator is prepared by the steps of: The method comprises the following steps: According to the operation steps in S1, the 37-degree Celsius water bath is melted for 2-4 hours, and the centrifugation is performed at 4-degree Celsius and 4000 revolutions per minute for 10 minutes.

4. The method of claim 1, wherein the D-dimer and FDP calibrator is prepared by the steps of: The method comprises the following steps: According to the operation steps in S3, the full-automatic coagulation instrument is model CX-9000.

5. A method for preparing a D-dimer and FDP composite quality control product according to claim 1, characterized in that, The method comprises the following steps: According to the operation steps in S4, the termination solution is a termination plasma.

6. The method of claim 1, wherein the D-dimer and FDP calibrator is prepared by the steps of: The method comprises the following steps: According to the operation steps in S4, the termination solution contains 2.5% preservative 1, 0.05% preservative 2, and 5% protective agent.

7. A method for preparing a D-dimer and FDP composite quality control product according to claim 1, characterized in that, The method comprises the following steps: According to the operation steps in S4, the termination solution is added into the reaction solution in an equal volume, and the reaction is performed at room temperature for 5-30 minutes.

8. A method for preparing a D-dimer and FDP composite quality control product according to claim 1, characterized in that, The method comprises the following steps: According to the operation steps in S5, the quality control product is prepared according to the target content and proportion.

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