An APTT detection reagent, and a preparation method and application thereof

Abstract

An APTT detection reagent, a preparation method and application thereof. The APTT detection reagent comprises an R1 reagent and an R2 reagent, the R1 reagent comprises a phospholipid mixture selected from any two of phosphatidylserine, phosphatidylethanolamine and phosphatidylcholine; and the concentration of the phospholipid mixture ranges from 0.1 g / L to 1 g / L. The APTT detection reagent improves the sensitivity of the APTT reagent to LA patients and the APTT detection has improved thermal stability.

Description

An APTT detection reagent, its preparation method and application Technical Field

[0001] This article relates to biological detection technology, particularly the application and usage of a synthetic phospholipid reagent in the detection of activated partial thromboplastin time (APTT). Background Technology

[0002] Activated partial thromboplastin time (APTT) is an indicator used to evaluate blood coagulation. Because early experiments used kaolin and cephalin, this test was historically known as kaolin-cephalin clotting time (KCCT). APTT is achieved by activating coagulation factor XII with contact factor activators (such as kaolin, tannic acid, etc.) within a standard in vitro timeframe, replacing PF3 with partial thromboplastin (cephalin), and adding calcium... 2+ After activation of the intrinsic coagulation pathway, the time required for plasma coagulation is observed. Activated partial thromboplastin time (IPT) reflects the activity of coagulation factors I, II, V, VIII, IX, X, XI, and XII in plasma. It is a screening test to check for any dysfunction of the body's intrinsic coagulation system and is also used for monitoring oral heparin.

[0003] When lupus anticoagulant (LA) is present in the blood of subjects suspected of having hemophilia or coagulation factor abnormalities, it is impossible to determine whether the prolongation of coagulation time is due to the deficiency or abnormality of coagulation factors or to LA by using reagents for the conventional APTT assay.

[0004] Lupus anticoagulant (LA) is a phospholipid-dependent, pathological circulating anticoagulant that can be produced naturally in the body or due to autoimmunity. It is mostly IgG, with a minority being IgM or a mixture of both. Lupus anticoagulant (LA) prolongs phospholipid-dependent clotting time in in vitro tests and is named after its initial discovery in the serum of patients with systemic lupus erythematosus (SLE). It primarily prolongs phospholipid-dependent clotting time by binding to β2-glycoprotein I (β2-GPⅠ) and other negatively charged phospholipids. Lupus anticoagulant (LA) can be found in antiphospholipid syndrome (APS), SLE, connective tissue diseases, etc., and is closely related to arterial and venous thrombosis and pathological pregnancy in these patients.

[0005] Lupus anticoagulant (LA) is present in 1%–5% of healthy individuals and can increase with age, often showing a weak positive result without clinical manifestations of atopic dermatitis (APS). Infections, tumors, and certain medications can also cause a temporary increase in LA. Therefore, patients should undergo a repeat lupus anticoagulant test 12 weeks after the initial test to confirm the diagnosis. False positive results can also occur when patients are treated with anticoagulants such as heparin and warfarin.

[0006] Currently, there is no unified standard or reference value for LA testing, and the three guidelines on LA testing [International Society for Thrombosis and Haemostasis (ISTH) 2009, British Committee for Standards in Hematology (BCSH) 2012, and Clinical Laboratory Standards Institute (CLSI) 2014] are not entirely consistent.

[0007] Therefore, there remains a need for high-quality APTT assay reagents suitable for both general patients and those with LA-related conditions. Summary of the Invention

[0008] The following is an overview of the subject matter described in detail herein. This overview is not intended to limit the scope of the claims.

[0009] This application provides an APTT detection reagent, its preparation method, and its application. The APTT detection reagent improves the sensitivity of APTT reagents to LA patients and exhibits enhanced thermal stability.

[0010] In an embodiment of this application, an APTT detection reagent is provided. The APTT detection reagent may include reagent R1 and reagent R2. Reagent R1 includes an activator and a mixture of phospholipids selected from any two of phosphatidylserine, phosphatidylethanolamine, and phosphatidylcholine. The concentration range of the phospholipid mixture is 0.1 g / L to 1 g / L.

[0011] In some embodiments of this application, preferably, the phospholipid mixture may be a phospholipid mixture of phosphatidylethanolamine and phosphatidylcholine.

[0012] In some embodiments of this application, preferably, the phospholipid mixture can be a phospholipid mixture of phosphatidylethanolamine and phosphatidylserine.

[0013] In some embodiments of this application, preferably, the phospholipid mixture may be a phospholipid mixture of phosphatidylserine and phosphatidylcholine.

[0014] In some embodiments of this application, the weight ratio of the two phospholipids in the phospholipid mixture is in the range of 3:7-7:3.

[0015] Preferably, the weight ratio of phosphatidylserine to phosphatidylcholine in the phosphatidylserine and phosphatidylcholine phospholipid mixture is in the range of 3:7-7:3.

[0016] In some embodiments of this application, the R1 reagent may further include a buffer solution and a metal ion compound;

[0017] Preferably, the metal ion compound is selected from any one of the following: magnesium chloride, aluminum chloride, and copper sulfate; and the concentration range of the metal ion compound is 20 mg / L-50 mg / L.

[0018] In some embodiments of this application, the R1 reagent may optionally include one or more of surfactants, antioxidants, and stabilizers;

[0019] Preferably, the surfactant is selected from any one of the following: polyvinylpyrrolidone and polyethylene glycol, and has a degree of polymerization of 2000-20000; and the concentration range of the surfactant is 1g / L-8g / L.

[0020] Preferably, the antioxidant is selected from any one of the following: rosmarinic acid, butylated hydroxyanisole, and catechin; and the concentration range of the antioxidant is 1 mM-6 mM.

[0021] Preferably, the stabilizer is selected from any one or more of the following: glycine, mannitol, BSA, and Proclin 300; and the concentration range of the glycine is 5 g / L-20 g / L; the concentration range of the mannitol is 2 g / L-10 g / L; the concentration range of the BSA is 0 g / L-10 g / L; and the proportion of Proclin 300 is 0.15%.

[0022] In some embodiments of this application, the R1 reagent may include surfactants, antioxidants, and stabilizers;

[0023] Preferably, the surfactant is selected from any one of the following: polyvinylpyrrolidone and polyethylene glycol, and has a degree of polymerization of 2000-20000; and the concentration range of the surfactant is 1g / L-8g / L.

[0024] Preferably, the antioxidant is selected from any one of the following: rosmarinic acid, butylated hydroxyanisole, and catechin; and the concentration range of the antioxidant is 1 mM-6 mM.

[0025] Preferably, the stabilizer is selected from any one or more of the following: glycine, mannitol, BSA, and Proclin 300; and the concentration range of the glycine is 5 g / L-20 g / L; the concentration range of the mannitol is 2 g / L-10 g / L; the concentration range of the BSA is 0 g / L-10 g / L; and the proportion of Proclin 300 is 0.15%.

[0026] In some embodiments of this application, the R2 reagent may include a calcium salt;

[0027] Preferably, the calcium salt may be selected from the salt of calcium with an inorganic acid or an organic acid;

[0028] The calcium salt can be CaCl2; and the concentration of the CaCl2 can be 25 mM.

[0029] In some embodiments of this application, the R2 reagent may optionally include a protective agent.

[0030] In some embodiments of this application, the protective agent may optionally be Proclin300.

[0031] In some embodiments of this application, the buffer solution is selected from any one of the following: trihydroxyaminomethane buffer, piperazine-1,4-diethanesulfonic acid buffer, 4-hydroxyethylpiperazine ethanesulfonic acid buffer, 3-(N-morpholine)propanesulfonic acid buffer; and the concentration range of the buffer solution is 10mM-100mM, pH 7.0-9.0.

[0032] In some embodiments of this application, preferably, the buffer solution can be 4-hydroxyethylpiperazine ethanethioic acid buffer.

[0033] In some embodiments of this application, preferably, the buffer solution is a trihydroxyaminomethane buffer solution.

[0034] In some embodiments of this application, preferably, the buffer solution is piperazine-1,4-diethanesulfonic acid buffer.

[0035] In some embodiments of this application, preferably, the buffer solution can be 4-hydroxyethylpiperazine ethanesulfonic acid buffer.

[0036] In some embodiments of this application, preferably, the buffer solution is 3-(N-morpholine)propanesulfonic acid buffer.

[0037] In some embodiments of this application, the activator may be selected from any of the following: ellagic acid, kaolin, kaolin, diatomaceous earth, colloidal silica; and the concentration range of the activator is 20 mg / L-500 mg / L.

[0038] Preferably, the activator can be ellagic acid.

[0039] In some embodiments of this application, preferably, the activator can be kaolin.

[0040] In some embodiments of this application, the activator is preferably kaolin.

[0041] In some embodiments of this application, the activator is preferably diatomaceous earth.

[0042] In some embodiments of this application, the activator is preferably colloidal silicon dioxide.

[0043] In some embodiments of this application, the metal ion compound may be selected from any one of the following: magnesium chloride, aluminum chloride, copper sulfate; and the concentration range of the metal ion compound is 20 mg / L-50 mg / L.

[0044] In some embodiments of this application, preferably, the metal ion compound can be copper sulfate.

[0045] In some embodiments of this application, preferably, the copper sulfate is copper sulfate pentahydrate.

[0046] In some embodiments of this application, preferably, the metal ion compound can be magnesium chloride.

[0047] In some embodiments of this application, preferably, the metal ion compound can be aluminum chloride.

[0048] In some embodiments of this application, the surfactant may be selected from any one of the following: polyvinylpyrrolidone and polyethylene glycol, and the degree of polymerization is 2000-20000; and the concentration range of the surfactant is 1g / L-8g / L.

[0049] In some embodiments of this application, the surfactant may preferably be polyvinylpyrrolidone.

[0050] In some embodiments of this application, the surfactant is preferably polyethylene glycol.

[0051] In some embodiments of this application, the antioxidant may be selected from any one of the following: rosmarinic acid, butylated hydroxyanisole, catechin; and the concentration range of the antioxidant is 1 mM-6 mM.

[0052] In some embodiments of this application, preferably, the antioxidant can be rosmarinic acid.

[0053] In some embodiments of this application, preferably, the antioxidant can be butylated hydroxyanisole.

[0054] In some embodiments of this application, preferably, the antioxidant can be catechin.

[0055] In some embodiments of this application, the stabilizer may be selected from any one or more of the following: glycine, mannitol, bovine serum albumin (BSA), and Proclin 300; and the concentration range of the glycine is 5 g / L-20 g / L; the concentration range of the mannitol is 2 g / L-10 g / L; the concentration range of the BSA is 0 g / L-10 g / L; and the proportion of Proclin 300 is 0.15%.

[0056] In some embodiments of this application, the R1 reagent comprises 20-500 mg / L of activator, 0.1 g / L-1 g / L of phospholipid mixture, 10 mM-100 mM pH 7.0-9.0 buffer solution, and 20 mg / L-50 mg / L of metal ion compound.

[0057] In some embodiments of this application, the R1 reagent further includes 1 g / L-8 g / L surfactant, 1 mM-6 mM antioxidant, or 0 g / L-20 g / L stabilizer.

[0058] In some embodiments of this application, the stabilizer is preferably glycine.

[0059] In some embodiments of this application, the stabilizer is preferably mannitol.

[0060] In some embodiments of this application, the stabilizer is preferably BSA.

[0061] In the embodiments of this application, a method for preparing the above-mentioned APTT detection reagent is provided, the preparation method comprising the following steps:

[0062] Preparation of reagent R1:

[0063] Adjust the pH of the buffer solution to neutral;

[0064] Add an activator to the buffer solution;

[0065] Then add metal ion compounds;

[0066] Then add a phospholipid mixture of 0.1 g / L to 1 g / L;

[0067] Then, optionally, surfactants, antioxidants, or stabilizers are added;

[0068] Preparation of reagent R2:

[0069] Dissolve the calcium salt and adjust its concentration to 25 mM.

[0070] In some embodiments of the preparation method of this application, the phospholipid mixture is a phospholipid mixture of phosphatidylethanolamine and phosphatidylcholine.

[0071] In some embodiments of the preparation method of this application, preferably, the phospholipid mixture can be a phospholipid mixture of phosphatidylethanolamine and phosphatidylserine.

[0072] In some embodiments of the preparation method of this application, preferably, the phospholipid mixture can be a phospholipid mixture of phosphatidylserine and phosphatidylglycerol.

[0073] In some embodiments of the preparation method of this application, preferably, the phospholipid mixture can be a phospholipid mixture of phosphatidylethanolamine and phosphatidylglycerol.

[0074] In some embodiments of the preparation method of this application, preferably, the phospholipid mixture can be a phospholipid mixture of phosphatidylserine and phosphatidylglycerol.

[0075] In some embodiments of the preparation method of this application, preferably, the phospholipid mixture can be a phospholipid mixture of phosphatidylglycerol and phosphatidylcholine.

[0076] In some embodiments of the preparation method of this application, preferably, the phospholipid mixture can be a phospholipid mixture of phosphatidylserine and phosphatidylcholine.

[0077] Preferably, in the phospholipid mixture of phosphatidylserine and phosphatidylcholine, the weight ratio of phosphatidylserine to phosphatidylcholine is in the range of 3:7-7:3.

[0078] In some embodiments of the preparation method of this application, the buffer solution is selected from any one of the following: trihydroxyaminomethane buffer, piperazine-1,4-diethanesulfonic acid buffer, 4-hydroxyethylpiperazine ethanesulfonic acid buffer, 3-(N-morpholine)propanesulfonic acid buffer; and the concentration range of the buffer solution is 10mM-100mM, pH 7.0-9.0.

[0079] In some embodiments of the preparation method of this application, preferably, the buffer solution is 4-hydroxyethylpiperazine ethanethiosulfate buffer.

[0080] In some embodiments of the preparation method of this application, preferably, the buffer solution can be a trihydroxyaminomethane buffer solution.

[0081] In some embodiments of the preparation method of this application, preferably, the buffer solution can be piperazine-1,4-diethanesulfonic acid buffer solution.

[0082] In some embodiments of the preparation method of this application, preferably, the buffer solution can be 3-(N-morpholine)propanesulfonic acid buffer.

[0083] In some embodiments of the preparation method of this application, the activator is selected from any one of the following: ellagic acid, kaolin, kaolin, diatomaceous earth, colloidal silica; and the concentration range of the activator is 20 mg / L-500 mg / L.

[0084] In some embodiments of the preparation method of this application, the activator is ellagic acid.

[0085] In some embodiments of the preparation method of this application, preferably, the activator can be kaolin.

[0086] In some embodiments of the preparation method of this application, preferably, the activator can be kaolin.

[0087] In some embodiments of the preparation method of this application, preferably, the activator can be diatomaceous earth.

[0088] In some embodiments of the preparation method of this application, preferably, the activator can be colloidal silicon dioxide.

[0089] In some embodiments of the preparation method of this application, the metal ion compound is selected from any one of the following: magnesium chloride, calcium chloride, aluminum chloride, and copper sulfate; and the concentration range of the metal ion compound is 20 mg / L-50 mg / L.

[0090] In some embodiments of the preparation method of this application, preferably, the metal ion compound is copper sulfate.

[0091] In some embodiments of the preparation method of this application, preferably, the copper sulfate is copper sulfate pentahydrate.

[0092] In some embodiments of the preparation method of this application, preferably, the metal ion compound can be magnesium chloride.

[0093] In some embodiments of the preparation method of this application, preferably, the metal ion compound can be calcium chloride.

[0094] In some embodiments of the preparation method of this application, preferably, the metal ion compound can be aluminum chloride.

[0095] In some embodiments of the preparation method of this application, the surfactant is selected from any one of the following: polyvinylpyrrolidone and polyethylene glycol, and the degree of polymerization is 2000-20000; and the concentration range of the surfactant is 1g / L-8g / L.

[0096] In some embodiments of the preparation method of this application, preferably, the surfactant can be polyvinylpyrrolidone.

[0097] In some embodiments of the preparation method of this application, preferably, the surfactant can be polyethylene glycol.

[0098] In some embodiments of the preparation method of this application, the antioxidant is selected from any one of the following: rosmarinic acid, butylated hydroxyanisole, and catechin; and the concentration range of the antioxidant is 1 mM-6 mM.

[0099] In some embodiments of the preparation method of this application, preferably, the antioxidant can be rosmarinic acid.

[0100] In some embodiments of the preparation method of this application, preferably, the antioxidant can be butylated hydroxyanisole.

[0101] In some embodiments of the preparation method of this application, preferably, the antioxidant can be catechin.

[0102] In some embodiments of the preparation method of this application, the stabilizer is selected from any one or more of the following: glycine, mannitol, BSA, and Proclin 300; and the concentration range of the glycine is 5 g / L-20 g / L; the concentration range of the mannitol is 2 g / L-10 g / L; the concentration range of the BSA is 0 g / L-10 g / L; and the proportion of Proclin 300 is 0.15%.

[0103] In some embodiments of the preparation method of this application, the stabilizer is preferably glycine.

[0104] In some embodiments of the preparation method of this application, the stabilizer is preferably mannitol.

[0105] In some embodiments of the preparation method of this application, preferably, the stabilizer can be BSA.

[0106] In some embodiments of this application, the R2 reagent optionally further includes a protective agent.

[0107] In some embodiments of this application, the protective agent is optionally Proclin300.

[0108] In the embodiments of this application, the above-mentioned APTT detection reagent is provided for use in APTT detection.

[0109] In the embodiments of this application, the above-described application of APTT testing for subjects suspected of having LA and coagulation factor deficiency is provided.

[0110] Compared with related technologies, this application comprises a high concentration of phospholipid mixtures and has the following advantages:

[0111] The high-concentration phospholipid mixture used in this application for APTT assay reagents reduces the sensitivity of the reagent to LA patients without altering the clotting time of normal individuals. This helps clinicians to accurately assess patients' coagulation function, avoids misjudgment of LA patients' coagulation function, and maintains the sensitivity of the APTT reagent to factor VIII.

[0112] Other features and advantages of this application will be set forth in the following description, and will be apparent in part from the description, or may be learned by practicing the application. Other advantages of this application may be realized and obtained by means of the methods described in the description. Attached Figure Description

[0113] The accompanying drawings are used to provide an understanding of the technical solutions of this application and constitute a part of the specification. They are used together with the embodiments of this application to explain the technical solutions of this application and do not constitute a limitation on the technical solutions of this application.

[0114] Figure 1 shows the results of the sensitivity of the APTT reagents of Examples 2-7 to factor VIII.

[0115] Figure 2 shows the results of the sensitivity of the APTT reagents of Examples 17-19 to factor VIII. Detailed Implementation

[0116] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0117] In subjects suspected of having hemophilia or coagulation factor disorders, where lupus anticoagulant (LA) is present in the blood, conventional APTT assays cannot determine whether the prolonged clotting time is due to LA or a deficiency or abnormality of the coagulation factor. Therefore, there remains a need for high-concentration phospholipid reagents for APTT assays that improve reagent stability without altering clotting time in healthy individuals, reduce sensitivity in LA patients, and facilitate accurate assessment of coagulation function by clinicians, avoiding misjudgments of LA patients' coagulation function. This application provides a high-concentration phospholipid reagent with improved sensitivity and thermal stability for APTT testing, along with its usage method.

[0118] Example

[0119] Preparation of APTT R1: Accurately weigh 2.39 g of 4-hydroxyethylpiperazine ethanethiol and add it to a 1 L beaker. Add 400 mL of purified water and stir to dissolve. After complete dissolution, adjust the pH to 7.4 with sodium hydroxide solution. Add 15 mg of ellagic acid and dissolve completely. Weigh 12.5 mg of copper sulfate pentahydrate and add it to the mixing tank. Stir to dissolve and dissolve completely. Then, add 2 g / L polyethylene glycol, 5 g / L BSA, 0.15% ProClin 300, and 0.72 g / L catechin in sequence to the mixing tank, ensuring that each ingredient is fully dissolved before adding the next. After all ingredients are dissolved and the solution is clear, add the specific mixed phospholipids according to the concentration in the table below. After thorough mixing and complete dissolution, adjust the pH to 7.4 with sodium hydroxide solution, bring the volume to 500 mL, stir well, and filter. The filtrate is the liquid APTT R1 reagent.

[0120] Table 1. Phospholipid components and their contents selected in the comparative examples and embodiments.

[0121] Phospholipid Types: Phosphatidylserine (g / L), Phosphatidylcholine (g / L), Phosphatidylethanolamine (g / L), Phosphatidylglycerol (g / L) Comparative Example 1: 0.65 Comparative Example 2: 0.65 Comparative Example 3: 0.65 Comparative Example 4: 0.65 Example 1: 0.05 0.05 / / Example 2: 0.5 0.5 / / Example 3: 0.025 0.025 Example 4: 0.05 0.05 Example 5: 0.15 0.15 / / Example 6 / 0.325 / Example 7 / 0.5 / Example 8 / 0.6 / Example 9 / 0.05 / 0.05 / Example 10 / 0.5 / Example 11 / 0.05 / 0.05 / Example 12 / 0.5 / 0.5 / Example 13 / 0.05 / 0.05 / Example 14 / 0.5 / 0.5 / Example 15 / 0.05 / 0.05 / Example 16 / 0.5 / 0.5 surface

[0122] Evaluation of reagent appearance

[0123] The prepared APTT R1 reagent was placed at room temperature for 6 hours, and then the presence or absence of precipitation was observed visually. The results are shown in Table 2.

[0124] Table 2. Stability overview of APTT R1 reagent at room temperature

[0125] Examples of Precipitation Examples of Precipitation Comparative Example 1 - Example 7 - Comparative Example 2 - Example 8 + Comparative Example 3 - Example 9 - Comparative Example 4 - Example 10 - Example 1 - Example 11 - Example 2 - Example 12 - Example 3 - Example 13 - Example 4 - Example 14 - Example 5 - Example 15 - Example 6 - Example 16 - surface

[0126] +: Sediment present; -: No sediment present.

[0127] Analysis: Table 1 shows that mixing two high-concentration phospholipids in the range of 0.05 g / L to 1 g / L will not produce precipitation, but precipitation will occur when the phospholipid concentration is too high.

[0128] Evaluation of normal plasma measurements

[0129] The APTT R1 reagent prepared above was mixed with calcium chloride solution, and the plasma of 3 normal individuals was measured. The mean value was calculated, and the coagulation time should be ≤35s. The experimental results are shown in Table 3.

[0130] Table 3 APTT Quality Overview

[0131] Do the average values ​​of reagent group plasma 1, plasma 2, and plasma 3 meet the requirements? Comparative Example 1: 33.8, 32.2, 33.4, 33.13; Comparative Example 2: 31.9, 31.9, 32.1, 31.97; Comparative Example 3: 31.2, 32.3, 31.83; Comparative Example 4: 32.3, 33.5, 31.6, 32.47; Example 1: 32.30, 32.1, 31.40; Example 2: 31.9, 31.5, 33.2, 32.20; Example 3: 28.5, 27.2, 27.57; Example 4: 26.8, 32.5, 30.7, 30.00; Example 5: 27.7, 28.3, 27.7, 27.90; Example 6: Implementation. Example 62628.927.527.47 is Example 725.82828.627.47 is Example 929.228.82828.67 is Example 1025.427.72726.70 is Example 1129.432.632.831.60 is Example 1232.131.833.432.43 is Example 1326.428.828.127.77 is Example 142828.225.727.30 is Example 1529.830.730.230.23 is Example 1632.332.332.832.47 is surface

[0132] Analysis: APTT reagents prepared from a mixture of any two phospholipids selected from phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol, and phosphatidylcholine all meet the requirements for measuring normal human plasma.

[0133] Evaluation of LA sensitivity

[0134] 1) Sample measurement

[0135] Normal plasma and LA-positive plasma were measured. LA-positive plasma and normal plasma (Coag Control N) were mixed 1:1, and the coagulation time was measured. Each sample was measured twice, and the average coagulation time was obtained. Commercially available normal plasma Coag Control N was used as a control for calculating the Rosner index.

[0136] 2) APTT measurement

[0137] APTT was measured using a Sysmex CS5100 fully automated coagulation analyzer. The specific method was as follows: 50 μL of sample was placed in a reaction vessel, and 50 μL of the APTT assay reagent was added and mixed. The mixture was incubated at 37°C for 3 min, followed by the addition of 50 μL of 25 mM calcium chloride solution. The mixture was immediately shaken and stirred until homogeneous. The time required for plasma coagulation was then measured. To obtain the LA sensitivity index of the reagent, the Rosner index was calculated based on the method of Rosner et al. The measurement results for each sample are shown in Table 3.

[0138] Rosner index = [(APTT measurement of 1:1 mixed plasma - APTT measurement of normal plasma) / APTT measurement of suspected LA-containing plasma] × 100%

[0139] Table 4 shows the sensitivity of the APTT reagent to LA in the comparative examples and the case studies.

[0140]

[0141] Analysis: Table 4 shows that APTT reagents formulated using a single phospholipid type have high sensitivity to LA. APTT reagents formulated with mixtures of phosphatidylserine + phosphatidylcholine, phosphatidylserine + phosphatidylethanolamine, phosphatidylglycerol + phosphatidylserine, phosphatidylglycerol + phosphatidylethanolamine, and phosphatidylglycerol + phosphatidylcholine show decreased Rosner index values ​​and lower sensitivity to LA. APTT reagents formulated with a mixture of phosphatidylcholine and phosphatidylethanolamine have even lower sensitivity to LA (data from Example 8 is not shown due to precipitation).

[0142] The results from Examples 3 to 6 show that, when using an APTT reagent prepared with a mixture of phosphatidylcholine and phosphatidylethanolamine phospholipids within a phospholipid concentration range of 0.1 g / L to 1 g / L, the APTT and Rosner index values ​​of LA-containing plasma decreased with increasing phospholipid types and concentrations. This demonstrates that the APTT assay reagent of the present invention can reduce the sensitivity of the reagent to LA without altering the clotting time in normal individuals.

[0143] Preparation of APTT R1: Accurately weigh 2.39 g of 4-hydroxyethylpiperazine ethanethiol and add it to a 1 L beaker. Add 400 mL of purified water and stir to dissolve. After complete dissolution, adjust the pH to 7.4 with sodium hydroxide solution. Add 15 mg of ellagic acid and dissolve completely. Weigh 12.5 mg of copper sulfate pentahydrate and add it to the mixing tank. Stir to dissolve and dissolve completely. Then, add 2 g / L polyethylene glycol, 5 g / L BSA, 0.15% ProClin 300, and 0.72 g / L catechin in sequence to the mixing tank, ensuring that each ingredient is fully dissolved before adding the next. After all ingredients are dissolved and the solution is clear, add the specific mixed phospholipids according to the concentration in the table below. After thorough mixing and complete dissolution, adjust the pH to 7.4 with sodium hydroxide solution, bring the volume to 500 mL, stir well, and filter. The filtrate is the liquid APTT R1 reagent.

[0144] Table 5 Phospholipid components and their contents in APTT R1 reagent

[0145] Examples: Phospholipids (g / L), Phosphatidylcholine (PC) (g / L), Phosphatidylethanolamine (PE) (g / L) Example 1: 70.65, 0.2, 0.45 Example 18: 0.65, 0.325, 0.325 Example 19: 0.65, 0.45, 0.2 Example 20: 10.3, 0.7 Example 21: 10.5, 0.5 Example 22: 10.7, 0.3 surface

[0146] Evaluation of reagent appearance

[0147] The prepared APTT R1 reagent was placed at room temperature for 6 hours, and then the presence or absence of precipitation was observed visually. The results are shown in Table 6.

[0148] Table 6. Stability overview of APTT R1 reagent at room temperature

[0149] Examples of precipitation: Examples 17-18-19-20-21-22 surface

[0150] +: Sediment present; -: No sediment present.

[0151] Analysis: Table 6 shows that no precipitation occurred during the storage of reagents in Examples 17 to 22.

[0152] Evaluation of LA sensitivity

[0153] 1) Sample measurement

[0154] Normal plasma and LA-positive plasma were measured. LA-positive plasma and normal plasma (Coag Control N) were mixed 1:1, and the coagulation time was measured. Each sample was measured twice, and the average coagulation time was obtained. Commercially available normal plasma Coag Control N was used as a control for calculating the Rosner index.

[0155] 2) APTT measurement

[0156] APTT was measured using a Sysmex CS5100 fully automated coagulation analyzer. The specific method was as follows: 50 μL of sample was placed in a reaction vessel, and 50 μL of the APTT assay reagent was added and mixed. The mixture was incubated at 37°C for 3 min. Then, 50 μL of 25 mM calcium chloride solution was added, and the mixture was immediately shaken to mix. The time required for plasma coagulation was then measured. To obtain the LA sensitivity index of the reagent, the Rosner index was calculated based on the method of Rosner et al.

[0157] Rosner index = [(APTT measurement of 1:1 mixed plasma - APTT measurement of normal plasma) / APTT measurement of suspected LA-containing plasma] × 100%

[0158] The results of each sample measurement are shown in Table 7.

[0159] Table 7 Sensitivity of APTT reagent to LA

[0160]

[0161] Analysis: As shown in Table 7, changing the mixing ratio of phospholipids can still reduce the sensitivity of LA, and the sensitivity of Examples 17 and 20 is relatively low.

[0162] Evaluation of thermal stability

[0163] The APTT reagents R1 and R2 from Examples 17 and 20 were divided into 12 equal portions. Six portions were stored at 2–8°C as controls, and the other six portions were placed at 37°C for accelerated stability assessment. The experiment lasted for 7 days. The Bio-Rad III level quality control was measured using a Sysmex CS5100 fully automated coagulation analyzer according to the instrument's instruction manual. Each sample was measured three times, and the mean, deviation 1, deviation 2, and deviation 3 were calculated.

[0164] Deviation 1 (%) = (Measurement result on day i of 2℃~8℃ - Measurement result on day 0 of 2℃~8℃) / (Test result on day 0 of 2℃~8℃) × 100% (i = 1, 2, ..., 6, 7);

[0165] Deviation 2 (%) = (Test result on day i at 37℃ - Test result on day 0 at 37℃) / (Test result on day 0 at 37℃) × 100% (i = 1, 2, ..., 6, 7);

[0166] Deviation 3 (%) = (Measurement result on day i at 37℃ - 2℃ to 8℃ on day i) / (Measurement result on day i at 2℃ to 8℃) × 100% (i = 0, 1, 2, ..., 6, 7)

[0167] The results are shown in Table 8.

[0168]

[0169]

[0170] The above results indicate that increasing the concentration of PE is beneficial to improving stability. Specifically, using phosphatidylethanolamine and phosphatidylcholine in a concentration ratio of 0.45:0.2 or 0.7:0.3 to prepare a high-concentration mixture of the two phospholipids for APTT reagent can significantly improve the stability of the reagent detection. The reagent does not precipitate during storage and is insensitive to LA.

[0171] Endogenous factor sensitivity test

[0172] Following the CLSI H47-A2 guidelines, commercially available factor-deficient plasma (name: Factor VIII deficient Plasma; manufacturer: Haematologic Technologies Inc.; catalog number: FVIII-ID) was mixed with normal human plasma to obtain diluted plasma with factor levels ranging from 0% to 100%. Factor VIII activity was measured using a Siemens coagulation factor activity assay kit. APTT reagents were prepared for each sample for testing. A line graph was plotted with coagulation factor activity on the x-axis and APTT test results of plasma at different concentrations of coagulation factor on the y-axis.

[0173] Using 35s as the dividing point, draw a straight line perpendicular to the Y-axis. The concentration of the coagulation factor corresponding to the point where the line intersects the line graph is the sensitivity concentration of the APTT reagent for that coagulation factor.

[0174] Table 9. Sensitivity of APTT reagent to factor VIII

[0175]

[0176] Analysis based on experimental data:

[0177] The above experimental results show (see Figure 1) that, in Examples 2-7, for factor VIII, when the APTT result is at the upper limit of the reference range, the corresponding coagulation factor VIII concentration is within the range of 30% to 45%, and the factor VIII is relatively sensitive.

[0178] Table 10: Data from the completed Factor VIII sensitivity analysis.

[0179]

[0180] The above experimental results show that, in Examples 2-7, for Factor VIII, when the APTT result is at the upper limit of the reference range, the corresponding coagulation Factor VIII concentration is within the range of 30% to 45%, and the factor is relatively sensitive to Factor VIII.

[0181] Table 11 Sensitivity of APTT reagent to factor VIII

[0182] VⅢ Factor Concentration % Example 17 Example 18 Example 194.859.255.558.510.449.347.247.019.642.340.641.130.036.836.837.338.233.135.134.849.731.831.829.860.030.630.029.970.728.428.228.679.427.127.327.493.026.426.326.999.027.526.827.4 surface

[0183] Analysis based on experimental data:

[0184] Table 12 Data from the completed Factor VIII sensitivity analysis

[0185]

[0186] The above experimental results show (see Figure 2) that, in Examples 17-19, for factor VIII, when the APTT result is at the upper limit of the reference range, the concentration of the corresponding coagulation factor VIII is within the range of 30% to 45%, and the factor VIII is relatively sensitive.

Claims

1. An APTT detection reagent, wherein, The APTT assay reagent includes reagent R1 and reagent R2. Reagent R1 includes an activator and a phospholipid mixture composed of phosphatidylethanolamine and phosphatidylcholine, wherein the weight ratio of the two phospholipids in the phospholipid mixture is in the range of 4.5:2 to 7:3; and the concentration range of the phospholipid mixture is 0.65 g / L to 1 g / L. Reagent R1 also includes a surfactant, an antioxidant, and a stabilizer; the antioxidant is selected from any one of the following: rosmarinic acid, butylated hydroxyanisole, and catechin; and the concentration range of the antioxidant is 1 mM to 6 mM.

2. The APTT detection reagent according to claim 1, wherein, The R1 reagent also includes a buffer solution and a metal ion compound.

3. The APTT detection reagent according to claim 2, wherein, The metal ion compound is selected from any one of the following: magnesium chloride, aluminum chloride, and copper sulfate; and the concentration range of the metal ion compound is 20 mg / L to 50 mg / L.

4. The APTT detection reagent according to claim 1, wherein, The surfactant is selected from any one of the following: polyvinylpyrrolidone and polyethylene glycol, and has a degree of polymerization of 2000-20000; and the concentration range of the surfactant is 1g / L-8g / L.

5. The APTT detection reagent according to claim 1, wherein, The stabilizer is selected from any one or more of the following: glycine, mannitol, BSA, and Proclin 300; and the concentration range of the glycine is 5 g / L-20 g / L; the concentration range of the mannitol is 2 g / L-10 g / L; the concentration range of the BSA is 0 g / L-10 g / L; and the proportion of Proclin 300 is 0.15%.

6. The APTT detection reagent according to any one of claims 1 to 5, wherein, The R2 reagent includes a calcium salt.

7. The APTT detection reagent according to claim 6, wherein, The calcium salt is selected from the salt of calcium with an inorganic acid or an organic acid.

8. The APTT detection reagent according to claim 6, wherein, The calcium salt is CaCl2; and the concentration of the CaCl2 is 25 mM.

9. The APTT detection reagent according to claim 2, wherein, The activator is selected from any one of the following: ellagic acid, kaolin, kaolin, diatomaceous earth, colloidal silica; and the concentration range of the activator is 20 mg / L-500 mg / L.

10. The APTT detection reagent according to claim 9, wherein, The activator is ellagic acid.

11. A method for preparing the APTT detection reagent according to any one of claims 4-10, wherein the preparation method comprises the following steps: Preparation of reagent R1: Adjust the pH of the buffer solution to neutral; Add an activator to the buffer solution; Then add metal ion compounds; Then add the phospholipid mixture; Then, surfactants, antioxidants, and stabilizers are added; R2 reagent is prepared by dissolving the calcium salt and making its concentration 25 mM.

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