Method and reagent for detecting fibrinogen content of whole blood or plasma

Abstract

The invention belongs to the technical field of biomedical examination and determination, and particularly relates to a method and a reagent for detecting the fibrinogen content of whole blood or plasma. The reagent comprises a buffer solution, inorganic salt, amino acid, thrombin, a stabilizer, a thrombin inhibitor, a blood clot enhancer, a heparin substance inhibitor and a preservative; the concentration of the thrombin is 5 U / mL to 200 U / mL; the thrombin inhibitor is one or more of argatroban, hirudin, lepiludine, bivalirudin, dextran sulfate and benzamidine hydrochloride; the blood clot reinforcing agent is one or more of tranexamic acid, aminomethylbenzoic acid, aminocaproic acid, alpha2-AP, TAFI, alpha2-M, PAI-1, PAI-2, glycoprotein rich in histidine (HRG) and C1-INH. According to the application, the reaction time of thrombin and fibrinogen is prolonged through the thrombin inhibitor so as to realize detection of whole blood and plasma samples, and meanwhile, the blood clot enhancement can be effectively increased through the blood clot enhancer without influencing the detection of fibrinogen.

Description

Technical Field

[0001] The present application relates to the technical field of biomedical testing and determination, and in particular to a method and reagent for detecting the content of fibrinogen in whole blood or plasma. Background Art

[0002] Fibrinogen (Fib) is the first coagulation factor of the human body. Fibrinogen is a key coagulation protein synthesized by liver cells, which plays a core role in blood coagulation and wound healing. As the most abundant coagulation factor in plasma, fibrinogen participates in the formation of thrombus and is an indispensable component in the hemostasis process. In addition, fibrinogen is also an acute reaction protein, and its plasma concentration increases in tissue damage, inflammation, infection, etc., so it is often used as a biomarker for disease prediction and diagnosis. Clinically, the determination of fibrinogen content is of great significance for assessing the risk of thrombosis in patients, monitoring the effect of treatment, and evaluating the synthetic function of the liver. Abnormal increase or decrease in fibrinogen levels is associated with a variety of diseases, including cardiovascular and cerebrovascular diseases, diabetes, hypertension, etc., making it an important indicator for assessing the risk of these diseases.

[0003] The fibrinogen content can usually be detected by a method based on the coagulable activity of fibrinogen, which is also the most commonly used method at present. In this method, by adding a high concentration of thrombin, the fibrinogen in the blood sample to be tested is converted into fibrin. The generated fibrin cross-links with each other, and then networks blood cells to form a blood clot. The blood clot adheres to the broken part of the blood vessel wall to prevent blood loss, and then the coagulation time is measured. The coagulation time is negatively correlated with the concentration of fibrinogen. The concentration of fibrinogen in the blood sample to be tested can be calculated through the standard curve. This method is simple, fast, and suitable for routine testing.

[0004] Currently, the blood sample to be tested is usually centrifuged plasma, which requires the blood sample to be collected first, centrifuged and then tested on a blood coagulation instrument. Plasma testing can be carried out in batches and the test results are accurate, but the testing process is long and the time required for testing is increased.

[0005] The use of whole blood samples to detect fibrinogen solves the problem of long detection time in plasma testing. Products that use whole blood samples are usually POCT products. After collecting the blood specimen, the sample does not need to be centrifuged and is directly added to the sample loading position of the reaction carrier. For example, the description of U.S. Patent 6448024B1 includes a sample loading hole and a flow channel coated with a thrombin reagent. After the sample is added to the sample loading hole, the sample will flow along the reaction flow channel. As fibrin is generated, the flow rate of the blood slows down. By detecting the position where the sample flow stops, the concentration of fibrinogen in the blood can be detected.

[0006] The advantage of whole blood sample fibrinogen detection is that the detection cycle is short. The shortest time from sample collection to result is only tens of seconds. Therefore, it is very suitable for occasions with high requirements for result time, such as operating room, emergency laboratory, clinical department, ICU, hemodialysis, etc. However, it also has some disadvantages, including: 1. It cannot be tested in large quantities at one time; 2. Because whole blood samples are used, the fibrinogen test results need to be calibrated with hematocrit; 3. The linear range is narrow. The required detection range of fibrinogen is at least 1-6g / L, while the linear range of existing products is relatively low. For example, the qLabs Fib product launched by Stago has a reportable range of 1-4g / L; 4. The coefficient of variation CV is large. Compared with the test of plasma samples, it is more susceptible to interference from various substances in the blood, such as heparin, triglycerides, etc.

[0007] Therefore, the present application proposes a method and reagent suitable for detecting the fibrinogen content in whole blood and plasma. Summary of the invention

[0008] In response to the above technical problems, the present application provides a method and reagent for detecting the fibrinogen content in whole blood or plasma.

[0009] According to a first aspect of one or more embodiments of the present application, a reagent for detecting whole blood or plasma fibrinogen is provided, the reagent comprising a buffer, an inorganic salt, an amino acid, thrombin, a stabilizer, a thrombin inhibitor, a clot enhancer, a heparin inhibitor, and a preservative; the concentration of the thrombin is 5U / mL to 200U / mL;

[0010] The thrombin inhibitor is one or more of argatroban, hirudin, lepirudin, bivalirudin, dextran sulfate, and benzamidine hydrochloride;

[0011] The blood clot enhancer is one or more of tranexamic acid, aminomethylbenzoic acid, aminocaproic acid, α2-AP, TAFI, α2-M, PAI-1, PAI-2, histidine-rich glycoprotein (HRG), and C1-INH.

[0012] Preferably, the buffer is one of glycine buffer, phthalic acid buffer, citrate buffer, phosphate buffer, borax buffer, barbital buffer, tris buffer, boric acid buffer, carbonate buffer, and hydroxyethylpiperazineethanesulfonic acid buffer.

[0013] Preferably, when the buffer is hydroxyethylpiperazineethanesulfonic acid buffer, the concentration of the hydroxyethylpiperazineethanesulfonic acid buffer is 10 mmol / L to 200 mmol / L.

[0014] Preferably, the inorganic salt is one or more of NaCl, CaCl2, calcium gluconate, and calcium lactate.

[0015] Preferably, the Ca in the reagent 2+ The concentration is 50mmol / L~200mmol / L.

[0016] Preferably, the amino acid is one or more of glycine, serine, lysine, arginine, alanine, and phenylalanine.

[0017] Preferably, the stabilizer is one of BSA and albumin.

[0018] Preferably, when the stabilizer is BSA, the content of BSA is 0.01% to 0.5%.

[0019] Preferably, the heparin inhibitor is one of polybrene and protamine sulfate, the concentration of the heparin inhibitor exceeds 0.1 mg / mL, and the heparin inhibitor can react with heparin to eliminate the influence of heparin substances.

[0020] Preferably, the preservative is one or more of PC300, PC950, BHA, BHT, and phenol.

[0021] According to a second aspect of one or more embodiments of the present application, a method for detecting whole blood or plasma fibrinogen is provided, the method using the reagent as described in the first aspect, the method comprising:

[0022] The sample to be tested is sucked into the microfluidic channel by a microfluidic pump, and the microfluidic channel is pre-coated and fixed with the reagent;

[0023] Shaking to dissolve the reagent and mix it with the sample to be tested;

[0024] The coagulation time of the sample to be tested is determined, and the content of fibrinogen in the sample to be tested is calculated according to the coagulation time.

[0025] Preferably, the reagent is fixed in the microfluidic channel by drying.

[0026] Preferably, the sample to be tested reacts with the reagent to generate fibrin to block the microchannel so that the sample to be tested coagulates.

[0027] Preferably, the coagulation time is determined according to the time when the microchannel is blocked.

[0028] The present invention prolongs the reaction time of thrombin and fibrinogen by using a thrombin inhibitor to achieve detection of plasma and whole blood samples. At the same time, a blood clot enhancer can effectively increase blood clot enhancement without affecting the detection of fibrinogen. This type of enhancer mainly acts on the fibrinolytic system, inhibits the action of the fibrinolytic system, thereby increasing the blood clot strength, and further improving the detection sensitivity of fibrinogen content. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The present application is further described below in conjunction with the accompanying drawings.

[0030] Figure 1 A diagram showing a detection range test result according to an exemplary embodiment of the present application;

[0031] Figure 2 An actual diagram showing the degree of blood clot coagulation according to an exemplary embodiment of the present application;

[0032] Figure 3 This is a flow chart of a method for detecting whole blood and plasma fibrinogen content, shown as an exemplary embodiment of the present application. DETAILED DESCRIPTION

[0033] The technical solutions in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present application.

[0034] In the description of the present application, it should be understood that the directions or positional relationships indicated by the terms "front", "rear", "left", "right", "up", "down", etc. are based on the directions or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific direction, be constructed and operated in a specific direction, and therefore should not be understood as a limitation on the present application.

[0035] like Figure 1-3 As shown, the present application provides a reagent for detecting whole blood or plasma fibrinogen, the reagent comprising a buffer, an inorganic salt, an amino acid, thrombin, a stabilizer, a thrombin inhibitor, a blood clot enhancer, a heparin inhibitor, and a preservative; the concentration of the thrombin is 5U / mL to 200U / mL;

[0036] The thrombin inhibitor is one or more of argatroban, hirudin, lepirudin, bivalirudin, dextran sulfate, and benzamidine hydrochloride;

[0037] The blood clot enhancer is one of tranexamic acid, aminomethylbenzoic acid, aminocaproic acid, α2-AP, TAFI, α2-M, PAI-1, PAI-2, histidine-rich glycoprotein (HRG), and C1-INH.

[0038] The target sample types of the present application include whole blood samples and plasma. After the whole blood sample (whether anticoagulated whole blood or non-anticoagulated whole blood) contacts with thrombin, coagulation will occur within a few seconds. Because the coagulation time is too short, it is difficult to accurately measure the content of fibrinogen. Therefore, it is necessary to extend the reaction time so that the coagulation time of fibrinogen of different concentrations is significantly different. The present invention uses a thrombin inhibitor to extend the reaction time, so that fibrinogen of different concentrations can be accurately distinguished.

[0039] Thrombin inhibitors can be used to inhibit the generation or activity of thrombin, thereby reaching the purpose of extending clotting time. The thrombin inhibitor used in the present application can be combined with thrombin to make thrombin lose the ability of shearing fibrinogen or reduce the function of shearing fibrinogen rate, such as one or more of argatroban, hirudin, lepirudin, bivalirudin, dextran sulfate, benzamidine hydrochloride. The higher the fibrinogen concentration, when competing with the above-mentioned substances for reacting thrombin, the shorter the time required for forming a blood clot, on the contrary, the lower the fibrinogen concentration, the longer the time required for forming a blood clot.

[0040] After using the above-mentioned thrombin inhibitors, the strength of the blood clot will decrease dramatically. This may be because the coagulation process will also activate the fibrinolytic system, which is an important physiological mechanism in the human body. Its function is to maintain the balance of blood circulation and ensure the fluidity of blood and the integrity of blood vessels. The main function of the fibrinolytic system is to dissolve the fibrin clot formed in the blood vessel. This process is called fibrinolysis (abbreviated as fibrinolysis). After the reaction time of thrombin and fibrinogen increases, the time given for blood clot dissolution also increases, so the strength of the blood clot is also reduced.

[0041] The present application has found that the use of a class of substances can effectively increase blood clot enhancement without affecting the detection of fibrinogen. This class of substances mainly acts on the fibrinolytic system, and increases the strength of blood clots by inhibiting the action of the fibrinolytic system. This class of substances includes tranexamic acid, aminobenzoic acid, aminocaproic acid, α2-AP, TAFI, α2-M, PAI-1, PAI-2, histidine-rich glycoprotein (HRG), C1-INH, and the like. Tranexamic acid, aminobenzoic acid, aminocaproic acid: This class of substances can form a stable complex with plasmin, blocking the activation of plasmin, thereby reducing the activity of the fibrinolytic system and reducing the blood's ability to dissolve blood clots; α2-AP: α2-antiplasmin (α2-Antiplasmin), which is a major fibrinolytic inhibitor, which inhibits the activity of plasmin by forming a complex with plasmin, thereby reducing the dissolution of blood clots and enhancing the stability of blood clots. PAI-1 & PAI-2: Plasminogen Activator Inhibitor-1 and Plasminogen Activator Inhibitor-2 (Plasminogen Activator Inhibitor), which are members of the serine protease inhibitor (serpin) superfamily, mainly inhibit tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA), thereby reducing fibrinolytic activity and enhancing the stability of blood clots. The concentrations required for different blood clot enhancers vary. The dosage of tranexamic acid, aminobenzoic acid, and aminocaproic acid is between 0.1% and 15%, and the dosage of α2-AP is between tens of umol / mL and tens of mmol / mL. TAFI: Thrombin-activated fibrinolysis inhibitor, its mechanism of action is to remove the lysine residues at the carboxyl terminus of fibrin, thereby reducing the formation of plasmin; HRG: A histidine-rich glycoprotein, HRG has a high binding affinity to the lysine binding site in the ring structure of the A chain of plasminogen, binds to plasminogen to form a reversible complex, and reduces the amount of plasminogen that can bind to fibrinolytic protein; C1-INH: C1 inhibitor, belongs to the family of serine protease inhibitors, is a single-chain glycoprotein containing 478 amino acids, which inhibits kallikrein, plasmin and FⅪa.

[0042] In a specific embodiment, the present application uses hydroxyethylpiperazineethanesulfonic acid (Hepes) as a buffer, calcium chloride mother solution as an inorganic salt, aminocaproic acid as a blood clot enhancer, and dextran sulfate as a thrombin inhibitor. The Fib buffer is prepared by mixing 2.00 ml of buffer + 4 ul of dextran sulfate + 0.2 g of aminocaproic acid + 20 ul of calcium chloride mother solution, and reagents 1 and 2 with thrombin concentrations of 40 U / mL and 60 U / mL after mixing are added to the thrombin mother solution, respectively. Whole blood samples and plasma samples are tested respectively to obtain the detection range of the reagents. Figure 1 As shown, the detectable fibrinogen concentration range can reach 0.5-10 g / L, and the coagulation time ranges from 19.1 to 63.6 s.

[0043] The precision of the test results is shown in Table 1, which lists the fibrinogen Fib content of different samples to be tested in Reagent 1 and Reagent 2, so as to verify the precision of the test results of the reagents in this application.

[0044] As shown in Table 1, the coefficient of variation CV of the Fib content of the reagent described in the present application is stable within 5%, with strong precision.

[0045] Table 1 Test results of the precision of Fib content in the detection reagent

[0046]

[0047] Table 2 lists the comparison of Fib test results of different types of samples to be tested. Plasma is the sample used in the standard test method. Compared with plasma, anticoagulated whole blood and non-anticoagulated whole blood have more blood cells. The ratio of blood cells to whole blood volume is usually called hematocrit, abbreviated as HCT. This value is generally between 10% and 70%. For the detection of whole blood samples (including anticoagulated whole blood and non-anticoagulated whole blood), HCT calibration is required. The detection reagent provided by the present invention can significantly prolong the coagulation time to cope with the detection of anticoagulated whole blood and non-anticoagulated whole blood, but in order to improve the consistency of the test results, it is necessary to recalibrate the relationship between the coagulation time and the fibrinogen content according to samples of different HCT. Accurately realize the detection of whole blood samples. Among them, in a specific embodiment, the conversion formula between coagulation time and fibrinogen content is Y=aX+b, and the conversion coefficients a and b of anticoagulated whole blood, non-anticoagulated whole blood, and plasma are different. a 抗凝全血 >a 血浆 >a 非抗凝全血 , b 抗凝全血 >b 非抗凝全血 >b 血浆 After calibrating the conversion coefficient, the Fib test results of different types of samples to be tested are shown in the following table. It can be found from the table that the reagent provided by the present invention can be applied to blood samples including anticoagulated whole blood, non-anticoagulated whole blood, and plasma.

[0048] Table 2 Comparison of Fib test results of different types of samples to be tested

[0049]

[0050] Experiments have shown that the degree of blood clot coagulation can be enhanced by adding tranexamic acid to the reagent. As shown in Table 3, increasing the amount of tranexamic acid added will increase the degree of sample coagulation. Figure 2 This is a practical example of the degree of blood clot coagulation in the present invention. Figure 2 (a) is fibrin filaments, Figure 2 (a) is a medium clot, Figure 2 (c) is a large clot.

[0051] Table 3 Effect of the amount of tranexamic acid TC added as a clot enhancer on coagulation results

[0052] serial number TC addition amount Sample 1 Sample 2 Sample 3 1 0.00％ Medium clot Fibrin Fibrin 2 1.50％ Medium to large clot Small and medium clots Fibrin 3 2.60％ Large clot Large clot Medium to large clot

[0053] Buffer plays a vital role in detection reagents. It is a solution composed of a weak acid and its conjugate base or a weak base and its conjugate acid. Its main function is to maintain the pH stability of the solution and resist the pH change caused by the addition of a small amount of external strong acid or strong base. Buffer ensures that biochemical experiments are carried out within a specific pH range, providing protection for enzyme activity and the stability of biomolecules. In addition, it also provides appropriate ionic strength, reduces nonspecific binding, and improves the specificity and accuracy of detection. In fields such as immunodiagnosis, buffers help promote immune responses and enhance the sensitivity and signal-to-noise ratio of detection. Buffers must also be compatible with other components in detection reagents to avoid causing unnecessary chemical reactions or interfering with test results. In terms of the preservation of biological samples, buffers can protect specific chemicals in the sample and maintain the structural integrity of the sample. In molecular biology techniques such as PCR, buffers control reaction conditions, including pH, ion concentration, and enzyme activity to ensure the effectiveness of the reaction. Therefore, the selection and preparation of buffers need to be precisely adjusted according to experimental requirements and reagent characteristics to ensure the accuracy and reliability of test results.

[0054] In a specific embodiment, the buffer contained in the reagent provided by the present application is one of glycine buffer, phthalic acid buffer, citrate buffer, phosphate buffer, borax buffer, barbital buffer, tris hydroxymethylaminomethane buffer, boric acid buffer, carbonate buffer, and hydroxyethylpiperazineethanesulfonic acid buffer. Preferably, hydroxyethylpiperazineethanesulfonic acid (Hepes) is an efficient zwitterionic buffer, which is widely used in the fields of biochemistry, molecular biology, and cell culture. Its pH buffer range is 6.8 to 8.2, especially in the range of pH 7.2 to 7.4, showing good buffering capacity. Hepes is non-toxic to cells, has good stability, has little change in decomposition constant at different temperatures, and is adapted to low temperature conditions. It can also reduce the cytotoxicity of chemicals and improve cell culture efficiency. However, it should be noted that Hepes may produce hydrogen peroxide under illumination, which is toxic to cells, so illumination should be avoided during operation. These characteristics make Hepes an indispensable buffer in scientific research and clinical applications.

[0055] In a specific embodiment, when the buffer is hydroxyethylpiperazineethanesulfonic acid buffer, the concentration of hydroxyethylpiperazineethanesulfonic acid buffer is 10 mmol / L to 200 mmol / L.

[0056] In a specific embodiment, the inorganic salt is one or more of NaCl, CaCl2, calcium gluconate, and calcium lactate. NaCl is added to the reagent components as an inorganic salt, and its main function is to maintain a certain ionic strength and keep the biologically active components in the reagent stable. The amount of NaCl added is generally between tens of mmol / L and hundreds of mmol / L.

[0057] Experiments show that adding Ca to the reagent 2+ It can increase the linear range of fibrinogen content detection and reduce the coefficient of variation CV. When the sample type to be tested is anticoagulated blood, CaCl2 needs to be added to the reagent to restore the ion Ca in the blood. 2+ When the sample type used is fresh non-anticoagulated whole blood, because the blood already contains Ca 2+ In a specific embodiment, preferably, CaCl2 should be added to the reagent. 2+ The concentration is adjusted and controlled at 50mmol / L~200mmol / L.

[0058] In a specific embodiment, the amino acid is one or more of glycine, serine, lysine, and arginine. Amino acids play two key roles as additives in reagents. First, they help maintain the stability of bioactive components in the blood, which is essential for ensuring that the reagents maintain their effectiveness during storage and use. Secondly, amino acids can also improve the re-dissolution efficiency of reagents, which means that they can be dissolved more quickly and more completely, thereby improving the efficiency and accuracy of the experiment. For example, amino acids such as glycine, serine, lysine, and arginine can all be added to reagents as stabilizers and cosolvents. They enhance the performance of the reagents by interacting with other components in the reagents, making them more reliable and efficient in the process of detecting fibrinogen content.

[0059] In a specific embodiment, the stabilizer is one of BSA and albumin. Bovine serum albumin (BSA) can be used as an enzyme stabilizer in biochemical experiments to prevent enzyme decomposition and nonspecific adsorption. It can also reduce the denaturation of some enzymes and reduce the denaturation caused by adverse environmental factors such as heating, surface tension and chemical factors. In addition, BSA can prevent the enzyme from being lost due to adsorption to the tube wall, thereby improving the efficiency and accuracy of the experiment.

[0060] In a specific embodiment, when the stabilizer is BSA, the mass fraction of BSA is 0.01% to 0.5%.

[0061] In a specific embodiment, the heparin inhibitor is polybrene, and the concentration of the heparin inhibitor exceeds 0.1 mg / mL, and the heparin inhibitor can react with heparin to eliminate the influence of heparin. In some specific departments or some people, such as operating rooms, the blood will contain heparin-like substances, which are used to quickly prolong the blood clotting time. Heparin substances can react with thrombin to reduce the content of thrombin that can participate in fibrinogen conversion, thereby affecting the clotting time of the reaction, so it is necessary to remove the influence of these substances. Heparin substances usually carry a negative charge, and heparin substances in the blood can be separated by neutralizing positive and negative charges. The abbreviation of polybrene is "Polybrene", and it also has another name called hexadimethrine bromide. Polybrene will be used as a preferred option for heparin inhibitors in this application.

[0062] After experimental demonstration, as shown in Table 4, 0.1 mg / L polybrene was selected as a heparin inhibitor to test the effects of different heparin concentrations on the Fib test results. By adding different amounts of heparin in a gradient, it was found that 0.1 mg / L polybrene could effectively resist heparin within a concentration of 1 U / mL and control the bias of the experimental results within 10%, indicating that heparin inhibitors can reduce the impact of heparin on fibrinogen test results and are credible.

[0063] Table 40.1 mg / L polybrene affects the test results of Fib at different heparin concentrations

[0064]

[0065] In a specific embodiment, the preservative is one of PC300, PC950, BHA, BHT, and phenol. The preservative is used to inhibit the growth of bacteria. The present application does not limit the type of preservative, and general preservatives can be used for the reagents proposed in the present application.

[0066] The present application also provides a method for detecting whole blood or plasma fibrinogen, such as Figure 3 As shown, the method uses the reagent described in the first aspect for detection, and the method comprises:

[0067] Step S1, sucking the sample to be tested into the microchannel by a microfluidic pump, the microchannel is pre-coated and fixed with the reagent;

[0068] Step S2, shaking to dissolve the reagent and mix it with the sample to be tested;

[0069] Step S3, determining the coagulation time of the sample to be tested, and calculating the content of fibrinogen in the sample to be tested according to the coagulation time.

[0070] In a specific embodiment, the reagent is fixed in the microfluidic channel by drying. The reagent is a dry reagent and can be stable for at least 12 months at room temperature.

[0071] In a specific embodiment, the sample to be tested reacts with the reagent to generate fibrin to block the microchannel and cause the sample to be tested to coagulate. Among them, the thrombin inhibitor can significantly prolong the time of detecting fibrinogen and increase the strength of the blood clot, so as to achieve the purpose of detecting the coagulation time in the entire interval.

[0072] In a specific embodiment, the coagulation time is determined according to the position where the microchannel is blocked. The generation of fibrin will slow down the flow of blood and gradually block the microchannel. By detecting the position where the sample flow stops, the concentration of fibrinogen in the sample can be detected.

[0073] The present invention prolongs the reaction time of thrombin and fibrinogen by using a thrombin inhibitor to achieve detection of whole blood samples. At the same time, the clot enhancer can effectively increase the clot enhancement without affecting the detection of fibrinogen. This type of enhancer mainly acts on the fibrinolytic system, inhibits the action of the fibrinolytic system, thereby increasing the strength of the clot to further improve the detection effect of the fibrinogen content. In addition, the fibrinogen concentration range that can be detected by the present invention can reach 0.5 to 10 g / L.

[0074] The reagents provided in this application can also be used to detect APTT, PT, and TT to meet different detection needs and increase the scope of application. Among them, APTT (activated partial thromboplastin time): This is a sensitive screening test for the endogenous coagulation system, which mainly reflects the activity of endogenous coagulation factors. The determination of APTT can be used to detect the levels of coagulation factors VIII, IX, XI, and XII in plasma, and has important applications in the diagnosis of hemophilia, coagulation factor deficiency, liver disease, and monitoring of heparin treatment. APTT prolongation may indicate an obstruction of the endogenous coagulation pathway, such as factor deficiency or the presence of anticoagulants. PT (prothrombin time): PT is an indicator of the function of the exogenous coagulation system, mainly reflecting the activity of coagulation factors I, II, V, VII, and X in plasma. It is a commonly used screening test for detecting whether there is any obstruction in the function of the exogenous coagulation system of the body, and is also an important monitoring indicator for clinical anticoagulant treatment. Prolongation of PT may be seen in congenital coagulation factor deficiency, liver disease, vitamin K deficiency, use of oral anticoagulants, etc. TT (thrombin time): TT refers to the time it takes for blood to coagulate after adding standardized thrombin to plasma, which mainly reflects the ability of fibrinogen to convert into fibrin. The extension of TT may indicate the presence of heparin or heparin-like substances, increased fibrinogen degradation products, low (no) fibrinogenemia, etc.

[0075] Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present application.

Claims

1. A reagent for detecting whole blood or plasma fibrinogen, characterized in that: The reagents include a buffer, an inorganic salt, an amino acid, thrombin, a stabilizer, a thrombin inhibitor, a blood clot enhancer, a heparin inhibitor, and a preservative; the concentration of the thrombin is 5 U / mL to 200 U / mL; The thrombin inhibitor is one or more of argatroban, hirudin, lepirudin, bivalirudin, dextran sulfate, and benzamidine hydrochloride; The blood clot enhancer is one or more of tranexamic acid, aminomethylbenzoic acid, aminocaproic acid, α2-AP, TAFI, α2-M, PAI-1, PAI-2, histidine-rich glycoprotein (HRG), and C1-INH.

2. The reagent according to claim 1, characterized in that The buffer is one of glycine buffer, phthalic acid buffer, citric acid buffer, phosphate buffer, borax buffer, barbital buffer, tris(hydroxymethylaminomethane) buffer, boric acid buffer, carbonate buffer and hydroxyethylpiperazineethanesulfonic acid buffer.

3. The reagent according to claim 2, characterized in that When the buffer is hydroxyethylpiperazineethanesulfonic acid buffer, the concentration of the hydroxyethylpiperazineethanesulfonic acid buffer is 10 mmol / L to 200 mmol / L.

4. The reagent according to claim 1, characterized in that The inorganic salt is one or more of NaCl, CaCl2, calcium gluconate and calcium lactate.

5. The reagent according to claim 4, characterized in that The Ca content of the reagent 2+ The concentration is 50mmol / L~200mmol / L.

6. The reagent according to claim 1, characterized in that The amino acid is one or more of glycine, serine, lysine, arginine, alanine and phenylalanine.

7. The reagent according to claim 1, characterized in that The stabilizer is one of BSA and albumin.

8. The reagent according to claim 7, characterized in that When the stabilizer is BSA, the mass fraction of BSA is 0.01% to 0.5%.

9. The reagent according to claim 1, characterized in that The heparin inhibitor is one of polybrene and protamine sulfate, the concentration of the heparin inhibitor exceeds 0.1 mg / mL, and the heparin inhibitor can react with heparin to eliminate the influence of heparin substances.

10. The reagent according to claim 1, characterized in that The preservative is one or more of PC300, PC950, BHA, BHT and phenol.

11. A method for detecting whole blood or plasma fibrinogen using the reagent according to claim 1, characterized in that: The method comprises: The sample to be tested is sucked into the microfluidic channel by a microfluidic pump, and the microfluidic channel is pre-coated and fixed with the reagent; Shaking to dissolve the reagent and mix it with the sample to be tested; The coagulation time of the sample to be tested is determined, and the content of fibrinogen in the sample to be tested is calculated according to the coagulation time.

12. The method for detecting whole blood or plasma fibrinogen according to claim 11, characterized in that: The reagents are fixed in the microfluidic channels by drying.

13. The method for detecting whole blood or plasma fibrinogen according to claim 11, characterized in that: The sample to be tested reacts with the reagent to generate fibrin which blocks the microchannel and causes the sample to be tested to coagulate.

14. The method for detecting whole blood or plasma fibrinogen according to claim 13, characterized in that: The coagulation time is determined according to the time when the microchannel is blocked.

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