Use of sodium thiocyanate in the preparation of a kit for the detection of coagulation items
By adding sodium thiocyanate to the coagulation test kit, the problem of insufficient accuracy of domestic coagulation test kits has been solved, and the accuracy and clinical relevance of various coagulation test methods have been improved. It is applicable to coagulation method, chromogenic substrate method and immunoturbidimetric method.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- WUHAN EASYDIAGNOSIS BIOMEDICINE
- Filing Date
- 2023-06-16
- Publication Date
- 2026-05-26
Smart Images
Figure CN116819104B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of coagulation testing technology, specifically relating to a kit for coagulation testing, which contains sodium thiocyanate and can significantly optimize the clinical comparison results of the kit. Background Technology
[0002] Coagulation is the complex process by which blood changes from a liquid to a gel-like state, involving the regulation and interaction of coagulation factors, thrombin, fibrinogen, and the fibrinolytic system. Coagulation tests can assist in disease diagnosis, assess surgical risks, or monitor the use of anticoagulants. Coagulation tests include prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), fibrinogen (FIB), fibrin(ogen) degradation products (FDP), D-dimer, antithrombin III (AT-III), and anti-Xa.
[0003] Currently, there are three main methods used for coagulation testing: coagulation method, chromogenic substrate method, and immunoturbidimetric method. The coagulation method uses optical transmission + percentage endpoint method to detect blood clotting time. The light source is an LED lamp; filtered light shines on the sample, and a photodiode receives the transmitted light signal and converts it into an electrical signal. After A / D conversion, software processes the data and plots a coagulation curve. The percentage endpoint method is used to determine the clotting time. Coagulation items that can be detected by the coagulation method include prothrombin time, activated partial thromboplastin time, thrombin time, and fibrinogen. The immunoturbidimetric method involves coating the antibody corresponding to the analyte onto uniformly sized latex particles. When the corresponding antigen and antibody bind, a complex is formed. The increase in the volume of the complex causes a change in transmitted light, i.e., a change in absorbance. The content of the detected substance is then calculated from the change in absorbance. Coagulation items that can be detected by immunoturbidimetric method include fibrin(ogen) degradation products and D-dimer. Chromogenic substrate methods infer the content and activity of a analyte by measuring changes in the absorbance of the chromogenic substrate. The principle involves the artificial synthesis of a compound that can be catalytically cleaved by enzymes, with the compound linked to a chromogenic substance. During detection, the chromogenic substance dissociates, causing a color change in the sample. The intensity of the color is proportional to the analyte and directly proportional to the absorbance value, thus allowing for precise quantification of the analyte. Coagulation parameters that can be detected using chromogenic substrate methods include antithrombin III and anti-Xa.
[0004] The coagulation market is currently experiencing good growth, but the major manufacturers in the domestic market are all importers, represented by Sysmex, Stago, and Werfen, which together hold 90% of the domestic market share. The overall quality of domestic coagulation reagent kits is still in its early stages of development, and the accuracy and relevance of the reagents need further improvement. Based on the current market environment, this invention is proposed. Summary of the Invention
[0005] In view of this, the present invention aims to provide a kit for coagulation test, specifically by adding sodium thiocyanate (CAS No. 540-72-7) to significantly improve the clinical relevance of the coagulation test kit.
[0006] The specific technical solution of the present invention is as follows:
[0007] A kit for detecting coagulation parameters, containing sodium thiocyanate; the kit is specifically based on the principle of any one of the following methods: coagulation method, chromogenic substrate method, and immunoturbidimetric method for detecting coagulation parameters. Specifically, the coagulation parameters of the present invention include prothrombin time, activated partial thromboplastin time, thrombin time, fibrinogen, fibrin(ogen) degradation products, D-dimer, antithrombin III, and anti-Xa.
[0008] During their research, the inventors discovered that adding sodium thiocyanate during coagulation testing using the coagulation method can improve the clinical relevance of the test results and increase the accuracy of the reagents. Further extending this method to other coagulation testing methods, they found that this technique is universally applicable across the three mainstream coagulation testing methodologies (coagulation method, chromogenic substrate method, and immunoturbidimetric method), effectively improving the clinical relevance of the reagents and demonstrating strong versatility.
[0009] Furthermore, when the above kit is specifically designed for detecting coagulation using a coagulation method, the final concentration of sodium thiocyanate in the detection system is 20-600 mmol / L.
[0010] The coagulation method kit mentioned above also includes a coagulation reagent, such as tissue thromboplastin suspension, bovine thrombin, or rabbit brain phospholipids. The final concentration of the tissue thromboplastin suspension in the detection system is preferably 0.1-5%, the final concentration of the bovine thrombin in the detection system is preferably 5-50 IU / mL, and the final concentration of the rabbit brain phospholipids in the detection system is preferably 0.1-0.5%, by mass percentage.
[0011] The above-mentioned coagulation method kit may further include at least one of the following: buffer solution, stabilizer, surfactant, and preservative.
[0012] The buffer solution is specifically HEPES buffer, MES buffer or Tris buffer, with a pH of 5-9 and a preferred concentration of 50-150 mmol / L.
[0013] Stabilizers include at least one of inorganic salts, macromolecular proteins, carbohydrates, and amino acids; more specifically,
[0014] The inorganic salt is at least one of sodium chloride, calcium chloride, and ammonium sulfate, and the final concentration of the inorganic salt in the detection system is preferably 0.2-2%, more preferably 0.2-1.5%, by mass percentage;
[0015] The macromolecular protein is at least one of BSA, gelatin and xanthan gum, and the final concentration of the macromolecular protein in the detection system is preferably 0.1-5%, more preferably 0.1-3%, by mass percentage;
[0016] The sugar is at least one of sucrose, trehalose and glucose, and the final concentration of the sugar in the detection system is preferably 0.5-5%, more preferably 0.5-3%, by mass percentage;
[0017] The amino acid includes at least one of glycine, histidine, and lysine, and the final concentration of the amino acid in the detection system is preferably 10-100 mmol / L, more preferably 15-50 mmol / L.
[0018] The surfactant is at least one of Tween 20, Tween 80, Triton X-100, Triton X-405 and Kao A90, and the final concentration of the surfactant in the detection system is 0.1-0.5% by mass percentage.
[0019] The preservatives are Proclin 300 and / or sodium azide, and their final concentration in the test system is 0.1-0.5% by mass percentage.
[0020] It is understandable that in the above coagulation method kit, each reagent component can be stored separately, or it can be directly pre-prepared into a detection system without the sample to be tested.
[0021] Furthermore, when the above kit is specifically designed for detecting coagulation using a chromogenic substrate method, the kit contains reagent A1 and reagent A2, wherein reagent A1 contains sodium thiocyanate with a final concentration of 20-150 mmol / L, and reagent A2 contains a chromogenic substrate.
[0022] In the above-mentioned chromogenic substrate method kit, reagent A1 also includes at least one of the following: buffer, stabilizer, preservative, and protein preparation.
[0023] The buffer solution is HEPES buffer or Tris buffer, with a pH of 5.5-8.5 and a preferred concentration of 50-150 mmol / L.
[0024] Stabilizers include at least one of inorganic salts, macromolecular proteins, sugars, and amino acids; more specifically:
[0025] The inorganic salt is at least one of sodium chloride, calcium chloride and ammonium sulfate, and the final concentration of the inorganic salt in reagent A1 is preferably 0.2-2%, more preferably 0.2-1.5%, by mass percentage;
[0026] The macromolecular protein is at least one of BSA, gelatin and xanthan gum, and the final concentration of the macromolecular protein in reagent A1 is preferably 0.1-5%, more preferably 0.1-3%, by mass percentage;
[0027] The sugar is at least one of sucrose, trehalose and glucose, and the final concentration of the sugar in reagent A1 is preferably 0.5-5%, more preferably 0.5-3%, by mass percentage;
[0028] The amino acid is at least one of glycine, histidine, and lysine, and the final concentration of the amino acid in reagent A1 is preferably 10-100 mmol / L, more preferably 15-50 mmol / L.
[0029] The preservative is Proclin 300 and / or sodium azide, and the final concentration of the preservative in reagent A1 is preferably 0.1-0.5% by mass percentage.
[0030] The protein preparation is thrombin and / or activating factor X, wherein the final concentration of thrombin in reagent A1 is preferably 5-30 IU / mL, and the final concentration of activating factor X in reagent A1 is preferably 0.01-0.5 IU / mL.
[0031] Furthermore, when the above kit is specifically designed for the detection of coagulation items based on immunoturbidimetric assay, the kit includes reagent B1 and reagent B2, wherein reagent B1 contains sodium thiocyanate with a final concentration of 100-600 mmol / L, and reagent B2 includes antibody-coated latex microspheres.
[0032] In the above-mentioned immunoturbidimetric assay kit, reagent B1 also includes at least one buffer, stabilizer, surfactant, and preservative. Specifically:
[0033] The buffer solution is HEPES buffer, borate-sodium borate buffer or Tris buffer, with a pH of 5-9 and a preferred concentration of 10-200 mmol / L;
[0034] Stabilizers include at least one of inorganic salts, macromolecular proteins, and carbohydrates, and more specifically:
[0035] The inorganic salt is sodium chloride and / or calcium chloride, and the final concentration of the inorganic salt in reagent B1 is preferably 0.2-2% by mass percentage;
[0036] The macromolecular protein is BSA, and the final concentration of BSA in reagent B1 is preferably 0.1-5%, more preferably 0.1-3%, by mass percentage;
[0037] The sugar is at least one of sucrose, trehalose and glucose, and the final concentration of the sugar in reagent B1 is preferably 0.5-5%, more preferably 0.5-3%, by mass percentage.
[0038] The surfactant is at least one of Tween 20, Tween 80, Triton X-100, Triton X-405 and Kao A90, and the final concentration of the surfactant in the detection system is 0.1-0.5% by mass percentage.
[0039] The preservatives are Proclin 300 and / or sodium azide, and their final concentration in the test system is 0.1-0.5% by mass percentage.
[0040] Compared with the prior art, the beneficial technical effects of the present invention are as follows:
[0041] This invention significantly improves the clinical relevance of coagulation test results and enhances reagent accuracy by adding sodium thiocyanate to coagulation test reagents. Furthermore, this invention is universally applicable across mainstream coagulation testing methods, with a wide range of applications. Specifically, experimental data shows that in coagulation method test kits, sodium thiocyanate affects the process of fibrinogen converting into insoluble fibrin during the detection process, thus affecting the measured values of the test reagents. In chromogenic substrate method test kits, sodium thiocyanate enhances the protein interaction between antithrombin and thrombin or factor Xa, thereby improving specificity. In chromogenic substrate method test kits, sodium thiocyanate facilitates the formation of a hydrophobic layer during antigen-antibody complex formation, thereby improving reaction specificity. In addition, for chromogenic substrate method test kits, adding sodium thiocyanate not only improves clinical relevance but also significantly improves the linearity of the test reagents. Simultaneously, for immunoturbidimetric test kits, adding sodium thiocyanate effectively improves low-end sensitivity, thus significantly improving precision for lower values. Attached Figure Description
[0042] Figure 1 This is a comparison chart of the solidification times detected by the experimental reagent and the control reagent in Example 1;
[0043] Figure 2 This is a comparison graph showing the solidification time of the control group reagent and the comparison reagent in Example 1;
[0044] Figure 3 This is a comparison chart of the detection results of the experimental group reagent and the comparison reagent in Example 2;
[0045] Figure 4 This is a comparison chart of the detection results of the control group reagent and the comparison reagent in Example 2;
[0046] Figure 5 The graph shows the linearity test results of the reagents in the experimental group in Example 2;
[0047] Figure 6 This is a graph showing the linearity test results of the control group reagent in Example 2;
[0048] Figure 7 This is a comparison chart of the detection results of the experimental group reagent and the comparison reagent in Example 3;
[0049] Figure 8 This is a comparison chart of the detection results of the control group reagent and the comparison reagent in Example 3. Detailed Implementation
[0050] To better understand this invention, the embodiments of the invention are further described below with reference to one specific example each of the coagulation method, the chromogenic substrate method, and the immunoturbidimetric method, along with accompanying drawings. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the invention.
[0051] Unless otherwise specified, all reagents and materials used are commercially available.
[0052] Example 1
[0053] This example uses the coagulation method to detect prothrombin time (specifically, the time required for blood to come into contact with the coagulation reagent until prothrombin is activated and causes a blood sample to form a clot). An experimental group (containing sodium thiocyanate) and a control group (not containing sodium thiocyanate) were set up. The composition of the detection system for each group is shown in Table 1.
[0054] Table 1
[0055]
[0056] The reagents prepared for the experimental and control groups were tested using a fully automated coagulation analyzer (Sysmex CS2400) at a wavelength of 660 nm. 50 μL of sample was taken, and 100 μL of reagent was added. The reaction was carried out at 37 °C. The instrument set the transmitted light intensity immediately after the reagent was added, before coagulation, to 0%. As the coagulation reaction progressed, the transmitted light intensity gradually increased until it stopped changing after the coagulation reaction, at which point it was set to 100%. A predetermined value was set for the 50% transmitted light intensity, and the corresponding time was taken as the coagulation time.
[0057] Forty clinical plasma samples were simultaneously tested using reagents prepared for both the experimental and control groups, along with commercially available coagulation methods. A scatter plot was created, with the x-axis representing the coagulation time of the experimental or control group and the y-axis representing the coagulation time of the commercially available kit. The correlation coefficient R was then calculated. 2 .
[0058] The results are shown in Figure 1 and Figure 2 The test data shows that, compared with the control group, the clinical comparability correlation R was significantly higher in the experimental group. 2 The value increased from 0.8931 to 0.9846, indicating that adding sodium thiocyanate to the kit components can improve the clinical relevance of the prothrombin time assay kit.
[0059] Example 2
[0060] This example uses the chromogenic substrate method to detect antithrombin III. The antithrombin III detection kit used in this example includes reagent 1 and reagent 2. In both the experimental group and the control group, reagent 2 is a chromogenic substrate dilution dissolved in MES buffer. The components of reagent 1 in the experimental group and the control group are shown in Table 2.
[0061] Table 2
[0062]
[0063] The clinical relevance of the experimental and control groups to commercially available products using the chromogenic substrate method was compared, and the linear range was tested. The procedure is as follows:
[0064] The reagents prepared for the experimental and control groups were tested using a fully automated coagulation analyzer (Sysmex CS2400) at a wavelength of 405 nm. 16 μL of sample or calibrator was added to 112 μL of diluent and reacted at 37°C for 40 s. 24 μL of the mixture was then added to 175 μL of reagent 1 and reacted at 37°C for 220 s. Finally, 33 μL of reagent 2 was added. Transmittance data at the set start and end times were analyzed, and the change in absorbance per minute was calculated using linear regression. Calibration was first performed using calibrators. A standard curve was plotted on a log-logarithmic coordinate system with antithrombin III concentration on the x-axis and the corresponding change in absorbance on the y-axis. The concentration of antithrombin III in the sample could be determined by comparing its absorbance change with the standard curve.
[0065] In this example, 40 clinical plasma samples were tested using both the control group reagent and the experimental group reagent, along with the commercially available product. A scatter plot was created, with the x-axis representing the concentration in the experimental or control group and the y-axis representing the concentration in the 40 clinical plasma samples tested using the commercially available kit. The plot is shown below. Figure 3 and Figure 4 As shown, the correlation R of the data is calculated. 2Linearity tests were performed on the control group and experimental group reagents in the examples. The horizontal axis represents the tested concentration, and the vertical axis represents the theoretical concentration of the experimental or control group. A scatter plot was then created, as shown in the figure. Figure 5 and Figure 6 As shown, the correlation R of the data is calculated. 2 .
[0066] The test data shows that, compared with the control group, the clinical comparability correlation R was significantly higher in the experimental group. 2 The value increased from 0.8906 to 0.9826, indicating that adding sodium thiocyanate to the kit components improved the clinical relevance of the antithrombin III assay reagent. Linearity increased from the experimental group R... 2 The linearity of the antithrombin III assay reagent was optimized by adding sodium thiocyanate to the kit components, from 0.9994 to 0.9884 in the control group.
[0067] Example 3
[0068] In this example, D-dimer was determined by immunoturbidimetric assay. The D-dimer assay kit used in this example includes reagent 1 and reagent 2. Reagent 2 is the same for both the experimental group and the control group, both of which contain antibody-coated latex microspheres. The specific components of reagent 1 for the experimental group and the control group are shown in Table 3.
[0069] Table 3
[0070]
[0071] The clinical relevance of the experimental and control groups was compared with that of commercially available products (immunoturbidimetric assay), and the precision in the low-value range was tested.
[0072] The process is as follows:
[0073] The reagents prepared for the experimental and control groups were tested using a fully automated coagulation analyzer (Sysmex CS2400) at a wavelength of 660 nm. 23 μL of sample or calibrator was added to 75 μL of reagent 1, and the mixture was reacted at 37°C for 30 seconds. Then, 75 μL of reagent 2 was added, and the reaction was repeated at 37°C for 90 seconds. Under the set conditions, the set start and end points maximized the absorbance change for each sample and achieved optimal approximate linearity. The transmitted light data between the two points was analyzed, and the absorbance change per minute was calculated using a linear regression equation. Calibration was first performed using calibrators, and a calibration curve was plotted with D-dimer concentration on the x-axis and the corresponding absorbance change on the y-axis. The concentration of D-dimer in the sample could be obtained by comparing its absorbance change with the calibration curve.
[0074] Forty clinical plasma samples were tested using the control group reagent and experimental group reagent in the examples, respectively, along with commercially available products. A scatter plot was created, with the horizontal axis representing the concentration in the experimental or control group and the vertical axis representing the concentration in the 40 clinical plasma samples tested using the commercially available kit. The plot is shown below. Figure 7 and Figure 8 As shown, the correlation R of the data is calculated. 2 Precision tests were performed on the control group reagents and experimental group reagents in the examples, and the coefficient of variation (CV) was calculated. The results are shown in Table 4.
[0075] Table 4
[0076] reagents 1 2 3 4 5 6 7 8 9 10 MEAN SD CV experimental group 0.48 0.52 0.49 0.48 0.47 0.50 0.51 0.54 0.52 0.48 0.50 0.02 4.57％ control group 0.42 0.49 0.56 0.57 0.62 0.41 0.50 0.46 0.49 0.61 0.51 0.07 14.47％
[0077] The test data shows that, compared with the control group, the clinical comparability correlation R was significantly higher in the experimental group. 2 The accuracy improved from 0.922 to 0.9883, indicating that adding sodium thiocyanate to the kit components improved the clinical relevance of the D-dimer assay reagent. The low-end precision CV decreased from 14.47% to 4.57%, demonstrating that adding sodium thiocyanate to the kit components optimized the low-end precision of the D-dimer assay reagent.
[0078] In conclusion, adding sodium thiocyanate to coagulation test kits can improve the clinical relevance of test results, and this method is applicable to coagulation methods, chromogenic substrate methods, and immunoturbidimetric methods.
[0079] The above embodiments are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be construed as limiting the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.
Claims
1. The application of sodium thiocyanate in the preparation of reagent kits for coagulation testing, characterized in that, The kit is based on the chromogenic substrate method for detecting coagulation parameters, including antithrombin III and anti-Xa. The kit includes reagent A1 and reagent A2, wherein reagent A1 contains sodium thiocyanate at a concentration of 20-150 mmol / L, and reagent A2 contains a chromogenic substrate.
2. The application according to claim 1, characterized in that, The reagent A1 further includes at least one of the following: buffer solution, stabilizer, preservative, and protein preparation; wherein, The buffer solution is HEPES buffer or Tris buffer, and the pH of the buffer solution is 5.5-8.5; The stabilizer includes at least one of inorganic salts, macromolecular proteins, sugars, and amino acids; The preservative is Proclin 300 and / or sodium azide; The protein preparation includes thrombin and / or activating factor X.
3. The application according to claim 2, characterized in that, The inorganic salt is at least one of sodium chloride, calcium chloride, and ammonium sulfate, and its concentration in reagent A1 is 0.2-2%; the macromolecular protein is at least one of BSA, gelatin, and xanthan gum, and its concentration in reagent A1 is 0.1-5%; the sugar is at least one of sucrose, trehalose, and glucose, and its concentration in reagent A1 is 0.5-5%; the amino acid is at least one of glycine, histidine, and lysine, and its concentration in reagent A1 is 10-100 mmol / L; the preservative has a concentration of 0.1-0.5% in reagent A1; the thrombin has a concentration of 5-30 IU / mL in reagent A1; and the activating factor X has a concentration of 0.01-0.5 IU / mL in reagent A1.