Single reagent, reagent card and method for rapidly detecting anti-Xa activity

Abstract

The invention relates to a single reagent for detecting anti-Xa activity and a corresponding reagent card. The single reagent is composed of 2-8 g / L of a buffer solution with low metal chelating ability, 0.1-0.5 U / mL of an activated Xa factor (FXa), 10-30 g / L of CaCl2 and a protective agent. The invention provides a brand-new method for detecting the anti-Xa activity, and the anti-Xa activity can be directly, accurately and quickly detected through the single reagent and the reagent card.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to a technology for detecting the activity of coagulation factors, in particular to a detection kit for the anti-Xa activity of heparin and low molecular weight heparin and a non-disease diagnosis detection method thereof. Background Art

[0002] Unfractionated heparin (UFH) and low-molecular-weight heparin (LMWH) are commonly used anticoagulants for the treatment or prevention of thromboembolism. UFH is often the drug of choice in settings such as hemodialysis, percutaneous transluminal angioplasty, and extracorporeal circulation. However, due to its unstable pharmacokinetics, individual anticoagulant efficacy varies widely. Adverse reactions include bleeding, thrombocytopenia, and osteoporosis with long-term use. The therapeutic response is poorly predictable, necessitating continuous dose adjustment. Therefore, to achieve optimal anticoagulant efficacy and reduce or avoid complications such as bleeding, monitoring is recommended during UFH therapy. LMWH, due to its long half-life and superior safety profile, is widely used for the prevention of deep vein thrombosis in orthopedic or general surgery, acute coronary syndrome, acute myocardial infarction, and anticoagulation during pregnancy. However, monitoring is also recommended for patients with obesity, renal insufficiency, children, and pregnancy.

[0003] Anti-Xa activity assays are specifically used to monitor the efficacy of unfractionated heparin, low-molecular-weight heparin, and novel oral anticoagulants, maximizing their anticoagulant effects while minimizing bleeding. Anti-Xa testing directly targets a specific coagulation factor, factor X, and offers a specific, targeted anticoagulant effect. Compared to the aPTT assay, it has fewer interfering factors across the entire intrinsic coagulation pathway, is internationally standardized, and provides stable and reliable results reflecting the therapeutic range. Over the past 30 years, other anticoagulants, including low-molecular-weight heparin, fondaparinux, direct thrombin inhibitors, and direct Xa anticoagulants, have been gradually approved and clinically used. Most institutions consider anti-Xa activity testing a more accurate, efficient, and reliable indicator of anticoagulation monitoring. Low-molecular-weight heparin (LWMH) is more common than unfractionated heparin, with hundreds of millions of units used annually. Anti-Xa activity assays are the only method for monitoring LMWH and fondaparinux sodium. Therefore, the development, implementation, and widespread use of anti-Xa testing programs are essential.

[0004] The technology disclosed in CN115219486A is the chromogenic substrate method, a mainstream anti-Xa activity detection method currently on the market. The principle of the chromogenic substrate method for determining heparin activity is to determine heparin activity based on the ability of the complex formed by AT III and heparin to neutralize activated Factor Xa. AT III in plasma and heparin form a complex (heparin-ATIII complex). A chromogenic substrate is first added to the plasma, followed by an excess of Factor Xa. When Factor Xa is added to the plasma-substrate mixture, two reactions occur simultaneously: hydrolysis of the substrate under the action of Factor Xa to release p-nitroaniline (pNA), and inhibition of Factor Xa by the heparin-AT III complex. After the competitive reactions reach equilibrium, the amount of pNA is inversely proportional to the heparin concentration in the reaction system. The heparin or low-molecular-weight heparin content can be calculated by establishing a standard curve. However, the chromogenic substrate method has the following drawbacks: 1) The chromogenic substrate method uses two reagents (chromogenic substrate and Factor Xa) and a diluent, and the two reagents can react with each other, which may cause cross-contamination, so the instrument carryover rate is required to be high;

[0005] 2) The chromogenic substrate needs to be stored away from light. It is unstable under light or temperature changes, which can easily affect the results.

[0006] 3) The chromogenic substrate method mainly detects the correspondence between color and concentration. Therefore, hemolysis, chylosing, etc. will interfere with the detection, and the sample is often unable to be diluted twice to reduce the interference.

[0007] 4) The chromogenic substrate method is often implemented on large coagulometers, which cannot achieve the purpose of timely detection. Heparin and low molecular weight heparin degrade rapidly, and have high requirements for timeliness.

[0008] 5) Currently, the anti-Xa detection kits available on the market are all multi-dose reagents. The reagents need to be refrigerated after opening and usually need to be used within half a month or a month.

[0009] CN114965459A discloses a dry chemical competitive inhibition chromogenic substrate method for detecting anti-Xa activity, wherein blood is collected from the patient's fingertip, placed in an anticoagulant tube, and then diluted, and finally the diluted sample is dripped onto a dry test strip for detection. The test strip's detection principle is as follows: red blood cells are filtered through the resin fiber upper layer of the dry chemical test strip, followed by plasma entering the middle layer to neutralize and inhibit the excess quantitative Xa. The plasma carries the remaining Xa from the reaction and permeates forward to the chromogenic substrate localization area, where it reacts with para-nitroaniline (PNA) to produce a yellow color development reaction within a specified time. When 405nm reflected light illuminates the chromogenic window (color development area), the incident light is reflected by the dark reflector on the bottom layer. The photosensor of the dry chemical detection instrument receives the sensed reflected light and converts it into an electrical signal for identification. Simultaneously, 600nm reflected light is used to eliminate interfering noise signals. After comparison with the calibration test, the instrument automatically generates and memorizes the calibration curve (which can also be made into a standard test strip) to calculate the quantitative anti-Xa activity content, enabling this test method to more accurately measure the activity content of heparin-like anticoagulants or substances. However, like existing methods, this method requires the use of a chromogenic substrate, which cannot avoid color interference in the sample and the stability of the chromogenic substrate. Summary of the Invention

[0010] Based on this, the purpose of the present invention is to provide a single reagent and detection method that can directly and rapidly detect anti-Xa activity.

[0011] The first aspect of the present invention provides a single reagent for detecting anti-Xa activity, which consists of 2-8 g / L of a buffer solution with low metal chelating ability, 0.1-0.5 U / mL of activated Xa factor (FXa), 10-30 g / L of CaCl2, and a protective agent.

[0012] To improve the high temperature resistance and storage performance of the reagent, a certain amount of a protective agent of suitable composition is added to the single reagent of the present invention. In some embodiments, the protective agent includes 10-80 g / L trehalose, 10-50 g / L glycine, 1-9 g / L sodium chloride, 1-10 g / L BSA, and 1-10 g / L 2-hydroxypropyl-β-cyclodextrin.

[0013] In some embodiments, the buffer with low metal chelation ability has a good buffering capacity for pH and does not affect the Ca ions in the reagent. Wherein, the buffer with low metal chelation ability can be one or more of tris-hydroxymethylaminomethane buffer (Tris-HCl), piperazine-1,4-diethanesulfonic acid buffer (PIPES), 3-morpholinepropanesulfonic acid (MOPS), and 4-hydroxyethylpiperazineethanesulfonic acid (HEPES); preferably, the buffer with low metal chelation ability is 2-8g / L 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), preferably 4-6g / L 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), and in some embodiments, the pH is preferably 6.0-8.0, and the pH is preferably 6.8.

[0014] Factor Xa is a key enzyme in the coagulation cascade, responsible for converting prothrombin to thrombin, which in turn converts fibrinogen to fibrin, forming a clot. This method assesses Factor Xa activity by measuring plasma clotting time. In some embodiments, the concentration of activated Factor Xa is 0.1 to 0.5 U / mL, preferably 0.1 to 0.3 U / mL, more preferably 0.18 to 0.22 U / mL, and most preferably 0.2 U / mL.

[0015] Commercially available reagents primarily consist of a three-reagent system consisting of activated Factor Xa, a Factor Xa chromogenic substrate, and a diluent. The present invention has discovered that a single reagent consisting of activated Factor Xa and CaCl2 can quickly obtain better test results. In some embodiments, the CaCl2 concentration is 10 to 30 g / L, preferably 18 to 24 g / L, and preferably 20 g / L. Since sodium citrate's anticoagulant mechanism is to complex calcium ions in the blood, preventing blood coagulation, adding an appropriate amount of CaCl2 to sodium citrate anticoagulated blood can directly initiate the reaction and obtain good test results.

[0016] In some preferred embodiments, the protective agent is 30-60 g / L trehalose, 15-30 g / L glycine, 3-7 g / L sodium chloride, 3-7 g / L BSA and 1-4 g / L 2-hydroxypropyl-β-cyclodextrin.

[0017] In some preferred embodiments, the protective agent is 40-50 g / L trehalose, 20-30 g / L glycine, 4-6 g / L sodium chloride, 4-6 g / L BSA and 1-3 g / L 2-hydroxypropyl-β-cyclodextrin.

[0018] In some preferred embodiments, the protective agent is 45-55 g / L trehalose, 20-30 g / L glycine, 4.5-5.5 g / L sodium chloride, 4.5-5.5 g / L BSA and 1.5-2.5 g / L 2-hydroxypropyl-β-cyclodextrin.

[0019] The second aspect of the present invention is to provide a reagent card for detecting anti-Xa activity, wherein the reagent card is obtained by dispensing any of the above-mentioned single reagents for detecting anti-Xa activity onto a test card for detecting anti-Xa activity and drying the resultant.

[0020] The third aspect of the present invention is to provide a method for detecting anti-Xa activity, comprising the following steps:

[0021] S1 obtains the plasma to be tested with sodium citrate as the anticoagulant;

[0022] S2: adding the plasma to be tested dropwise onto the test card;

[0023] S3 used an optical coagulometer to detect the concentrations of unfractionated heparin (UFH) and low molecular weight heparin (LMWH).

[0024] In some embodiments, the plasma to be tested is derived from humans, or mammals such as cows, sheep, dogs, and cats.

[0025] The present invention provides a new method for detecting anti-Xa activity, wherein the single reagent and reagent card can directly, quickly and accurately detect anti-Xa activity, which has the following advantages.

[0026] 1) The present invention abandons the multi-reagent approach and invents a single reagent for detecting anti-Xa activity. The main detection principle is that the reagent contains an appropriate amount of CaCl2 and activated Factor Xa. The CaCl2 is mainly used to neutralize the anticoagulant, allowing the blood to form a clot. AT III in the plasma sample and heparin form a complex (heparin-ATIII complex), which inhibits the activated Factor Xa in the reagent. The remaining activated Factor Xa activates the blood, ultimately forming a clot. Different concentrations of activated Factor Xa have a linear relationship with clotting time, so a standard curve can be established comparing clotting time with heparin and low-molecular-weight heparin concentrations.

[0027] 2) The reagent of this invention can be used in fully automated coagulometers, but is preferably used in optical coagulometers OCG-101 and OCG-102. Its main advantages are low production costs and simple manufacturing processes. Furthermore, the methodology primarily detects clot formation and is unaffected by sample color (e.g., bilirubin, hemoglobin, triglycerides, etc.).

[0028] 3) The optical coagulometers OCG-101 and OCG-102 used in this invention preferably use dry reagents, i.e., the reagents are dried on the bottom of the test card. This major advantage is that the test card can be individually packaged, used for a single test per card, and can be used for immediate testing and stored and transported at room temperature. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Bland-Altman analysis chart of unfractionated heparin. The horizontal axis represents the concentration distribution of unfractionated heparin clinical samples ranging from 0 to 2 U / mL, and the vertical axis represents the difference between the test results of the single reagent of the present invention and the control reagent. The control reagent is the test result of Haifeng reagent on Sysmex CS-2000i, and the self-developed test result refers to the test result of the single reagent of the present invention on OCG-101.

[0030] Figure 2 Bland-Altman analysis chart of low molecular weight heparin. The horizontal axis represents the concentration distribution of low molecular weight heparin clinical samples ranging from 0 to 2 U / mL, and the vertical axis represents the difference between the test results of the single reagent of the present invention and the test results of the control reagent. The control reagent is the test result of Haifeng reagent on Sysmex CS-2000i, and the self-developed test result refers to the test result of the single reagent of the present invention on OCG-101.

[0031] Figure 3 Comparison of unfractionated heparin clinical samples. The horizontal axis represents the test results of Haifeng reagent on Sysmex CS-2000i, and the vertical axis (Wondfo) represents the test results of the single reagent of the present invention on OCG-101.

[0032] Figure 4 Comparison of low molecular weight heparin clinical samples. The horizontal axis shows the results of the Haifeng reagent tested on the Sysmex CS-2000i. The vertical axis (Wondfo) shows the results of the single reagent of the present invention tested on the OCG-101. DETAILED DESCRIPTION

[0033] To facilitate understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the present disclosure more thorough and comprehensive.

[0034] Experimental procedures in the following examples, where specific conditions are not specified, generally followed conventional conditions, such as those in Molecular Cloning: A Laboratory Manual (4th edition, edited by Green and Sambrook, published in 2013), or according to manufacturer recommendations. All commonly used chemical reagents used in the examples were commercially available.

[0035] In the following examples, the experimental methods without specific conditions are generally carried out under conventional conditions or conditions recommended by the manufacturers. The various commonly used chemical reagents used in the examples are all commercially available products.

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which this invention pertains. The terms used in this specification are for the purpose of describing specific embodiments only and are not intended to limit the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0037] The following further describes the anti-Xa activity assay reagent and method provided by the present invention.

[0038] Example 1

[0039] The experiment was set up with 5 groups:

[0040] Prepare a single reagent for detecting anti-Xa activity: add 500 mg of 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) to 100 mL of pure water, stir for 10 minutes until the solid dissolves, and adjust the pH to 6.8 for use.

[0041] To 10 mL of the above buffer, add 200 mg of anhydrous CaCl2, 500 mg of trehalose, 250 mg of glycine, 50 mg of NaCl, 50 mg of BSA, and 20 mg of 2-hydroxypropyl-β-cyclodextrin. Continue stirring for 30 minutes until the solids dissolve. Add 1 mL of the above buffer to each of five centrifuge tubes. Then, add 1 U of activated Factor Xa (Group 1), 2 U of activated Factor Xa (Group 2), 3 U of activated Factor Xa (Group 3), 0.4 U of activated Factor Xa (Group 4), and 0.5 U of activated Factor Xa (Group 5) to each of the five groups, respectively. Stir for 10 minutes to mix thoroughly. Aliquot the prepared reagent and pipette 5 μL of the reagent onto the bottom of the test card. Dry the aliquoted test card in a 37°C oven for 1 hour. Install the dried reagent card onto the gear and perform the test with the semi-automatic coagulation analyzer OCG-101 / OCG-102.

[0042] The test card is commercially available. The OCG-101 / 102 test card used in this study is sold by Wondfo Biotech. The card consists of a plastic substrate and a gear. The reagent is applied to the reaction area of ​​the substrate. When the sample and reagent react to form a clot, the gear deflects it. The instrument records the time from the start of the test to the deflection of the clot, which is the clotting time. Activated Factor Xa was purchased from Ark Biosafety Technology (Guangzhou) Co., Ltd.

[0043] Normal plasma anticoagulant sodium citrate was used for testing. The mixed plasma was divided into 15 aliquots, each 1.0 mL. Five aliquots were then added with 0.0 U, 0.5 U, 1.0 U, 1.5 U, and 2.0 U of unfractionated heparin. Another five aliquots were then added with 0.0 U, 0.5 U, 1.0 U, 1.5 U, and 2.0 U of low-molecular-weight heparin. The remaining five aliquots were then added with 0 ng, 100 ng, 200 ng, 300 ng, and 450 ng of rivaroxaban. The prepared plasma was shaken for 10 minutes and then tested. A standard curve of clotting time and heparin concentration was plotted. The test results are shown in the figure below.

[0044] Table 1 Unfractionated heparin test results

[0045]

[0046] Table 2 Low molecular weight heparin test results

[0047]

[0048]

[0049] Table 3 Rivaroxaban test results

[0050]

[0051] The experimental results show that the five groups have a good correlation when testing the concentrations of unfractionated heparin, low molecular weight heparin and rivaroxaban. Among them, group 2 with an activated Xa factor of 0.2 U / mL has higher sensitivity, and the amount of activated Xa factor is preferably 0.1-0.3 U / mL.

[0052] Example 2

[0053] Two experimental groups were set up: 500 mg of 4-hydroxyethylpiperazine ethyl (HEPES) was added to 100 mL of pure water. Stir for 10 minutes until the solid dissolved, and the pH was adjusted to 6.8. To 10 mL of the above buffer, 0 mg of anhydrous CaCl2 (Group 1), 200 mg of anhydrous CaCl2 (Group 2), 500 mg of trehalose, 250 mg of glycine, 50 mg of NaCl, 50 mg of BSA, and 20 mg of 2-hydroxypropyl-β-cyclodextrin were added. Stirring was continued for 30 minutes until the solid dissolved. Then, 2.0 U of activated Factor Xa was added and stirred for 10 minutes to mix thoroughly. The prepared single reagent was aliquoted, and 5 μL of the single reagent was transferred to the bottom of the test card. The aliquoted test card was then dried in a 37°C oven for 1 hour. The dried reagent card was then mounted on the gear and ready for testing with the semi-automated hemagglutination analyzer OCG-101 / OCG-102.

[0054] The test was performed using normal human sodium citrate anticoagulated mixed plasma. The mixed plasma was divided into 10 1.0 mL portions. Five of the plasma portions were then added with 0.0 U, 0.5 U, 1.0 U, 1.5 U, and 2.0 U of unfractionated heparin, respectively. Another five plasma portions were then added with 0.0 U, 0.5 U, 1.0 U, 1.5 U, and 2.0 U of low molecular weight heparin, respectively. The prepared plasma was shaken for 10 minutes and then tested. A standard curve of clotting time and heparin concentration was constructed. The test results are shown in the table below.

[0055]

[0056] After research, it was found that an appropriate amount of CaCl2 must be added to the reagent, otherwise it cannot neutralize the anticoagulant in the blood collection tube, and ultimately the blood cannot coagulate.

[0057] Example 3: Stability test

[0058] Four sets of experiments were conducted: 500 mg of 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) was added to 100 mL of pure water. Stir for 10 minutes until the solid dissolved, then adjust the pH to 6.8. To 10 mL of the above buffer, 200 mg of anhydrous CaCl2, 500 mg of trehalose, and varying amounts of glycine, NaCl, BSA, and 2-hydroxypropyl-β-cyclodextrin were added. Stirring was continued for 30 minutes until the solid dissolved, followed by the addition of 1.0 U of activated Factor Xa and stirring for 10 minutes to mix thoroughly. The prepared reagent was aliquoted, and 5 μL of the reagent was transferred to the bottom of the test card. The aliquoted test card was then dried in a 37°C oven for 1 hour. The dried test card was then fitted with a gear for testing with Wondfo Biotech's OCG-101 / OCG-102.

[0059] 3.1 Different groups

[0060]

[0061] A test card with a single reagent was prepared according to the above embodiment. The reagent card was subjected to accelerated aging at 50°C for 2 weeks. During the two weeks, the test was performed using the same batch of reference plasma. The test was repeated three times at each time point. The mean and standard deviation of the samples were calculated. The results are as follows.

[0062] Group 1: High temperature stability results

[0063]

[0064] Group 2: High temperature stability test results

[0065]

[0066]

[0067] Group 3: High temperature stability test results

[0068]

[0069] Group 4: High temperature stability test results

[0070]

[0071]

[0072] As can be seen from the table above, after accelerated aging at 50°C for 14 days, only the deviation of the same sample in Group 2 was within ±10% for the reagent prepared according to Example 7. This indicates that the reagent has better stability and is suitable for storage at room temperature only after the addition of glycine, sodium chloride, and 2-hydroxypropyl-β-cyclodextrin.

[0073] Example 4 Clinical Comparison Experiment

[0074] The test card with a single reagent prepared according to the method of Group 2 of Example 1 (product of the present invention)

[0075] ) was used on the OCG-102 instrument to test 20 clinical samples with different concentrations of standard heparin and 20 clinical samples with different concentrations of low-molecular-weight heparin. Testing method: After preheating the test card to 37°C, add 20 μL of sample to obtain the corresponding clotting time. The heparin concentration in the sample was then determined based on a standard curve of heparin concentration and clotting time. The reference product was the gold standard, the Sysmex CS-2000i, and its accompanying heparin assay kit (chromogenic substrate method). The reference product was operated strictly according to its instructions, while the present invention was operated according to the OCG-102 operating instructions.

[0076] Unfractionated heparin test results (concentration: U / mL)

[0077]

[0078]

[0079] Low molecular weight heparin test results (unit: U / mL)

[0080]

[0081]

[0082] Figure 1 and Figure 3 , Figure 2 and Figure 4The results show that the single reagent, reagent card and method of the present invention have a better correlation with the commercially available reagents. When the reagent card of the present invention is used, most of the test data are

[0083] The test results are within the range of ±1.96SD, which means that they can provide an effective reference for clinical treatment.

[0084] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A single reagent for detecting anti-Xa activity, characterized in that: The single reagent consists of 2-8 g / L of a buffer solution with low metal chelating ability, 0.1-0.5 U / mL activated factor Xa (FXa), 10-30 g / L CaCl2, and a protective agent, wherein the protective agent includes 10-80 g / L of trehalose, 10-50 g / L of glycine, 1-9 g / L of sodium chloride, 1-10 g / L of BSA, and 1-10 g / L of 2-hydroxypropyl-β-cyclodextrin.

2. The single reagent for detecting anti-Xa activity according to claim 1, characterized in that The buffer solution is one or more of tris-hydroxymethylaminomethane buffer solution (Tris-HCl), piperazine-1,4-diethanesulfonic acid buffer solution (PIPES), 3-morpholinepropanesulfonic acid (MOPS), and 4-hydroxyethylpiperazineethanesulfonic acid (HEPES).

3. The single reagent for detecting anti-Xa activity according to claim 2, characterized in that The buffer solution is 2-8 g / L 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), preferably 4-6 g / L 4-hydroxyethylpiperazineethanesulfonic acid; and / or the pH of the buffer solution is 6.0-8.0, preferably 6.

8.

4. The single reagent for detecting anti-Xa activity according to claim 1, characterized in that The concentration of the activated factor Xa is preferably 0.1-0.3 U / mL, more preferably 0.18-0.22 U / mL.

5. The single reagent for detecting anti-Xa activity according to claim 1, characterized in that The CaCl2 concentration is 15-25 g / L, preferably 18-24 g / L, and more preferably 19-21 g / L.

6. The single reagent for detecting anti-Xa activity according to claim 1, characterized in that The protective agent comprises 30-60 g / L of trehalose, 15-30 g / L of glycine, 3-7 g / L of sodium chloride, 3-7 g / L of BSA and 1-4 g / L of 2-hydroxypropyl-β-cyclodextrin.

7. The single reagent for detecting anti-Xa activity according to claim 1, characterized in that The protective agent consists of 45-55 g / L of trehalose, 20-30 g / L of glycine, 4.5-5.5 g / L of sodium chloride, 4.5-5.5 g / L of BSA and 1.5-2.5 g / L of 2-hydroxypropyl-β-cyclodextrin.

8. A reagent card for detecting anti-Xa activity, characterized in that: The reagent card is obtained by dispensing the single reagent for detecting anti-Xa activity according to any one of claims 1 to 7 onto a test card for detecting anti-Xa activity and drying the resultant.

9. The reagent card according to claim 8, characterized in that The dispensing volume of the single reagent is 4-6uL, preferably 5uL.

10. A method for detecting anti-Xa activity, characterized in that: The following steps are involved: S1 obtains the plasma to be tested with sodium citrate as the anticoagulant; S2: adding the plasma to be tested dropwise onto the reagent card according to any one of claims 8 to 9; S3 used an optical coagulometer to detect the concentrations of unfractionated heparin (UFH) and low molecular weight heparin (LMWH).

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