Ultrasonic guided wave blood coagulation detection reagent card containing sensitive element and freeze-drying ball

Abstract

The invention discloses an ultrasonic guided wave blood coagulation detection reagent card comprising a sensitive element and a freeze-drying ball. Comprising a sample adding chamber, a reagent chamber and a detection channel which are sequentially communicated, a first freeze-drying ball and a second freeze-drying ball are placed in the reagent chamber, the first freeze-drying ball and the second freeze-drying ball are coated with an endogenous activating agent and an exogenous activating agent respectively, and the first freeze-drying ball and the second freeze-drying ball both contain a freeze-drying protective agent; a sensitive element is placed in the detection channel, and the sensitive element is made of a magnetostrictive material subjected to surface treatment; the ultrasonic guided wave blood coagulation detection reagent card reflects the viscosity change of a detection sample through the resonant frequency or amplitude change of the sensitive element. The ultrasonic guided-wave blood coagulation detection reagent card is not only suitable for trace sample detection, but also has the advantages of being rapid in detection, capable of being stored at normal temperature, easy and convenient to operate and the like.

Description

Technical Field

[0001] This invention relates to the field of coagulation testing, and more particularly to an ultrasonic guided wave coagulation test reagent card comprising a sensitive element and a lyophilized bulb. Background Technology

[0002] Thromboelastography (TEG) is an analyzer that comprehensively evaluates the coagulation and fibrinolysis processes. It requires no blood sample processing, using a small amount of whole blood to monitor platelets, coagulation factors, fibrinogen, the fibrinolytic system, and the interactions between other cellular components, accurately providing a patient's coagulation profile. It dynamically reflects the function of coagulation factors, fibrinogen, platelets, and fibrinolysis.

[0003] Invented by Hartert in 1948, TEG (platelet-fibrinogen esterase) began clinical application in the 1980s and has become an important clinical method for monitoring coagulation function. The advent of TEG is a milestone in personalized coagulation, enabling more rational application across departments. In today's context of increasingly scarce blood resources, its importance will only grow. Currently, routine coagulation function tests (such as PT and APTT) can only detect the activity of coagulation factors in plasma, reflecting a specific stage or coagulation product in the coagulation process. Platelets interact with coagulation factors during coagulation; coagulation tests without platelet participation cannot reflect the full picture of coagulation. TEG testing can comprehensively demonstrate the entire process of blood clot formation and development, from the activation of coagulation factors to the formation of a strong platelet-fibrin clot and then to fibrinolysis, showcasing the patient's overall coagulation status, the rate of clot formation, the strength of the clot, and the level of fibrinolysis. Rapid TEG simultaneously activates both intrinsic and extrinsic coagulation pathways in a shorter time, making it potentially more suitable for the diagnosis and real-time monitoring of coagulation abnormalities after trauma in the emergency department.

[0004] In a traditional thromboelastography (TEG) system, 0.34 mL of blood sample is added to a beaker, which is maintained at 37°C. The test cup containing the blood sample rotates uniformly at an angle of 4°45' and a speed of 1 revolution / 5s. During rotation, fibrin undergoes a polymerization reaction between the beaker wall and the torsion wire, initiating the coagulation process and gradually forming a blood clot. Once a thrombus forms, the metal probe placed in the blood sample test cup is subjected to the shear stress caused by the formation of the sample, resulting in left-right rotation. During this rotation, the metal needle generates an electric current by cutting magnetic lines of force. This current is processed by software, recorded on a computer, and plotted against time to create an image, which is the TEG curve.

[0005] However, the traditional method of detecting thromboelastography still has the following shortcomings:

[0006] In traditional TEG testing, the sample size is large, and the curve is easily affected by large air bubbles and table vibration during sample addition.

[0007] Traditional TEG instruments have issues with blood clots adhering to and detaching from the inner surface of the sample cup during routine thromboelastography testing; reagents need to be stored at 2–8°C. Summary of the Invention

[0008] To address the problem of blood clots adhering to and detaching from the inner surface of the sample cup in traditional TEG instrument testing, this invention provides an ultrasonic guided wave coagulation test kit containing a sensitive element and a lyophilized bulb. The ultrasonic guided wave coagulation test kit provided by this invention can utilize the sensitive element to detect changes in blood material properties, thereby obtaining a thromboelastography curve. Furthermore, this kit can be stored at room temperature, and coagulation testing can be completed with a mere 27 μL sample.

[0009] The technical solution adopted in this invention is:

[0010] I. An ultrasonic guided wave coagulation test reagent card containing a sensitive element and a lyophilized bulb

[0011] The reagent card includes a sample loading chamber, a reagent chamber, and a detection channel connected in sequence. The reagent chamber contains a first lyophilized bulb and a second lyophilized bulb. The first and second lyophilized bulbs are coated with an endogenous activator and an exogenous activator, respectively. Both the first and second lyophilized bulbs contain a lyophilization protectant. A sensitive element is placed in the detection channel. The sensitive element is made of a surface-treated magnetostrictive material. The ultrasonic guided wave coagulation test reagent card reflects the change in the viscosity of the test sample by the change in the resonant frequency or amplitude of the sensitive element under the combined action of a static magnetic field and a dynamic magnetic field.

[0012] Preferably, the magnetostrictive material includes, but is not limited to, permalloy, iron-cobalt-nickel alloy, pure nickel, and iron-based amorphous materials.

[0013] Preferably, the surface treatment is passivation or UV-cured coating.

[0014] Specifically, the sensitive element is an elongated structure, and the length direction of the elongated structure is parallel to the flow direction of the detection sample in the detection channel.

[0015] Specifically, the ultrasonic guided wave coagulation test reagent card mainly consists of a sample dispensing tube, a reagent tube, a test tube, a first sealing ring, and a second sealing ring;

[0016] Specifically, the main body of the sample dispensing tube is L-shaped. A sample dispensing chamber is formed in the vertical part of the sample dispensing tube, a reagent chamber is formed through the reagent tube, and a detection channel is formed through the detection tube. A first mounting hole is formed in the horizontal part of the sample dispensing tube, and a first sealing ring is coaxially arranged in the first mounting hole. One end of the reagent tube is inserted into the first mounting hole and abuts against the first sealing ring, thus achieving a detachable and sealed connection with the sample dispensing tube. A second mounting hole is formed at the other end of the reagent tube, and a second sealing ring is coaxially arranged in the second mounting hole. One end of the detection tube is inserted into the second mounting hole and abuts against the second sealing ring, thus achieving a detachable and sealed connection with the reagent tube. The other end of the detection tube is used to connect to an external negative pressure source during the detection process.

[0017] Preferably, the detection tube is made of a transparent or translucent material.

[0018] Preferably, the reagent chamber inside the reagent tube includes a reagent compartment and a reagent compartment outlet section. The reagent compartment contains a first lyophilized bulb and a second lyophilized bulb. The inner diameter of the reagent compartment outlet section is smaller than the inner diameter of the reagent compartment.

[0019] Preferably, the endogenous activator includes one or more combinations of ellagic acid, kaolin, diatomaceous earth, and kaolin; the exogenous activator is calcium chloride.

[0020] Preferably, the freeze-drying protectant is mainly composed of trehalose, PVP, bovine serum albumin and Proclin 300.

[0021] More preferably, the endogenous activator is kaolin, the concentration of kaolin before freeze-drying is 0.04 mg / mL, the concentration of calcium chloride before freeze-drying is 0.2 mol / L, and the mass-volume concentrations of trehalose, PVP, bovine serum albumin, and Proclin 300 before freeze-drying are 10%, 6%, 3%, and 0.05%, respectively.

[0022] Preferably, the ultrasonic guided wave coagulation test reagent card is stored in a double-sealed vacuum aluminum foil bag, and its shelf life is 2 years at room temperature.

[0023] Preferably, the first and second freeze-dried pellets are obtained through the following process: freezing the first and second solutions into pellets with a volume of 5 μL, and then freeze-drying them according to the following process to obtain the first and second freeze-dried pellets:

[0024] Pre-freeze at -43 to -47°C for 50 to 70 minutes;

[0025] Sublimation drying was carried out once at <10 Pa and -38 to -42 °C for 550 to 650 min.

[0026] Desorption and drying were carried out at <10 Pa and -37 to -33 °C for 50 to 70 minutes.

[0027] Desorption and drying were carried out under conditions of <10 Pa and -27 to -23 °C for 50 to 70 minutes;

[0028] Drying was carried out under conditions of <10 Pa and -17 to -13 °C for 50 to 70 minutes.

[0029] Drying was carried out under conditions of <10 Pa and -7 to -3 °C for 50 to 70 minutes.

[0030] Dry under conditions of <10 Pa and 3–7 °C for 50–70 min;

[0031] Desorption and drying were carried out at <10 Pa and 13-17℃ for 50-70 min;

[0032] Desorption and drying were carried out at <10 Pa and 23-27℃ for 300-400 min.

[0033] II. A method for coagulation detection using the ultrasonic guided wave coagulation test reagent card as described above.

[0034] The coagulation test method includes the following steps:

[0035] S1. Inject sodium citrate anticoagulated blood sample into the sample dispensing chamber of the ultrasonic guided wave coagulation test reagent card. The sodium citrate anticoagulated blood sample flows into the reagent chamber and reacts with the first lyophilized bulb and the second lyophilized bulb to form a test sample.

[0036] S2. Place the detection tube under a static-dynamic composite magnetic field. Drive the detection sample into the detection channel through negative pressure. The sensitive element in the detection channel undergoes ultrasonic vibration deformation under the combined action of the static and dynamic magnetic fields until the sensitive element is submerged in the detection sample.

[0037] Preferably, the detection tube is made of a transparent or semi-transparent material. In step S2, a color sensor is used to collect the color characteristics of the detection tube, thereby identifying the volume or liquid level of the sample in the detection tube, and thus determining whether the sensitive element is submerged in the sample.

[0038] S3. Real-time detection of the resonant frequency or amplitude of the sensitive element, and construction of a curve showing the change of resonant frequency or amplitude over time;

[0039] Preferably, in step S3, the detected main frequency includes the first, second, and third harmonics of the fundamental resonant frequency of the sensitive element.

[0040] S4. Generate a thromboelastography based on the curve of the resonant frequency or amplitude changing over time.

[0041] The beneficial effects of this invention are:

[0042] 1. The reagent card of this invention, through the use of sensitive elements and lyophilized bulbs, simultaneously solves the problem of blood clots adhering to and detaching from the inner surface of the sample cup in ordinary thromboelastography detection during the detection process. This not only shortens the time of ordinary thromboelastography coagulation detection, but also enables coagulation detection to be completed with a tiny sample of 27uL, thus improving accuracy, stability and reliability.

[0043] 2. The reagent card of this invention can be used with a fully enclosed micro-volume rapid ultrasonic guided wave coagulation elastography instrument to complete the test. During the test, you only need to insert the collected venous whole blood collection tube with sodium citrate anticoagulation into the reagent card to realize direct operation of whole blood. There is no need for complicated procedures such as manual sample addition. The results can be displayed after waiting for 10 minutes. The operation is simple and the test is fast.

[0044] 3. In the ultrasonic guided wave coagulation test reagent card of this invention, the lyophilized bulbs are spherical, round, and smooth in appearance, with low moisture content and good solubility. This avoids the impact of high moisture content on the stability of the lyophilized bulb concentration and improves the stability of the lyophilized bulb performance. This coagulation test reagent card can be stored at room temperature for 24 months. Compared with traditional kaolin liquid reagents on the market, which require storage at 2-8℃ and have high dry ice transportation costs, the preparation of lyophilized bulbs effectively solves the problems of short shelf life and cold chain transportation of existing similar reagents. Attached Figure Description

[0045] Figure 1 This is a schematic diagram of the reagent card structure in an embodiment of the present invention.

[0046] Figure 2 This is a schematic diagram of freeze-dried pellets in an embodiment of the present invention.

[0047] In the diagram: 1. Sample tube, 2. Reagent tube, 3. Detection tube, 4. First sealing ring, 5. Second sealing ring, 6. First lyophilized bulb, 7. Second lyophilized bulb. Detailed Implementation

[0048] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and technologies are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the art to which this invention pertains. The following definitions will apply for the purpose of interpreting this specification.

[0049] The "R value" described in this invention refers to the time from the beginning of coagulation to the start of fibrin formation. It represents the time required for thrombin and other coagulation factors to be fully activated and for fibrin to be generated. A prolonged R value indicates the use of anticoagulants or a deficiency of coagulation factors; a shortened R value indicates a hypercoagulable state of blood.

[0050] In this invention, the "K value" refers to the time required from the end of the R time period to the point where the tracing amplitude reaches 20 mm. It reflects the interaction between fibrin and platelets at the onset of clot formation, i.e., the rate of clot formation. The length of the K value is affected by the level of fibrinogen, and anticoagulants can prolong the K value.

[0051] The "MA value" described in this invention refers to the maximum strength of a fibrin / platelet clot, which is determined by the binding of fibrin and platelets through GPIIb / IIIa receptors. It is primarily influenced by two factors: fibrinogen and platelets. Platelets (approximately 80%) play a greater role than fibrinogen (approximately 20%). Abnormalities in platelet quality or quantity will affect the MA value.

[0052] The first aspect of the present invention provides an ultrasonic guided wave coagulation test reagent card comprising a sensitive element and a lyophilized bulb.

[0053] The ultrasonic guided wave coagulation test kit of this invention includes a sample application chamber, a reagent chamber, and a detection channel connected in sequence. The reagent chamber contains a first lyophilized bulb 6 and a second lyophilized bulb 7, which are coated with an endogenous activator and an exogenous activator, respectively. Both the first lyophilized bulb 6 and the second lyophilized bulb 7 contain a lyophilization protectant. A sensitive element is placed in the detection channel, and the sensitive element is made of a surface-treated magnetostrictive material. The isolated blood sample to be tested is mixed with the first lyophilized bulb 6 and the second lyophilized bulb 7 to form a test sample. The ultrasonic guided wave coagulation test kit reflects changes in the viscosity of the test sample through changes in the resonant frequency or amplitude of the sensitive element.

[0054] Specifically, the sensing element is a magnetostrictive material, including but not limited to permalloy, iron-cobalt-nickel alloy, pure nickel, and iron-based amorphous materials.

[0055] Preferably, the surface treatment methods include, but are not limited to, passivation methods, UV curing coating methods, etc.

[0056] Preferably, the sensing element has an elongated structure, and its shape includes, but is not limited to, a cylinder, a hollow cylinder, a U-shaped groove, a V-shaped groove, a rectangular sheet, a triangular sheet, and a rhomboid sheet.

[0057] Preferably, the length direction of the elongated structure is parallel to the flow direction of the sample in the detection channel.

[0058] Specifically, after surface treatment, magnetostrictive materials can avoid affecting the coagulation process, eliminating the need for additional inhibitory drugs such as abciximab. Furthermore, they can amplify the resonant signal, thereby reducing the required volume of ex vivo blood sample from the usual 300μL, 340μL, or 360μL to 27μL while maintaining the same detection accuracy. This not only saves on sample volume but also makes them suitable for applications such as finger-prick blood collection.

[0059] Specifically, such as Figure 1 As shown, the ultrasonic guided wave coagulation test kit mainly consists of a sample dispensing tube 1, a reagent tube 2, a detection tube 3, a first sealing ring 4, and a second sealing ring 5. The main body of the sample dispensing tube 1 is L-shaped, with the horizontal portion of the sample dispensing chamber, the reagent tube 2, and the detection tube 3 arranged coaxially in sequence. A sample dispensing chamber is located in the vertical portion of the sample dispensing tube 1. A reagent chamber is formed through the reagent tube 2, and a detection channel is formed through the detection tube 3. The upper inlet of the sample dispensing chamber serves as the sample inlet, the lower outlet of the sample dispensing chamber connects to the inlet of the reagent chamber, and the outlet of the reagent chamber connects to the inlet of the detection channel.

[0060] Specifically, the horizontal part of the sample tube 1 has a first mounting hole in the horizontal direction, which is connected to the sample dispensing chamber. A first sealing ring 4 is coaxially arranged in the first mounting hole. One end of the reagent tube 2 is inserted into the first mounting hole and abuts against the first sealing ring 4. The other end of the reagent tube 2 has a second mounting hole in the horizontal direction, which is coaxially arranged with a second sealing ring 5. One end of the detection tube 3 is inserted into the second mounting hole and abuts against the second sealing ring 5. The other end of the detection tube 3 is used to connect with an external negative pressure source during the detection process.

[0061] Preferably, the detection tube 3 is made of a transparent or semi-transparent material.

[0062] More preferably, the detection tube 3 is made of a transparent or semi-transparent flexible material.

[0063] Specifically, the reagent chamber within reagent tube 2 includes a reagent compartment and a reagent compartment outlet section. The reagent compartment holds a first lyophilized bulb 6 and a second lyophilized bulb 7. The inner diameter of the reagent compartment outlet section is smaller than the inner diameter of the reagent compartment. Simultaneously, the outlet of the sample loading chamber adopts a tapered shape. This tapered shape is coaxial with the first mounting hole and the reagent chamber. The inner diameter of the tapered shape gradually increases towards the reagent compartment, and its minimum inner diameter is smaller than the inner diameter of the reagent compartment. This ensures that the first lyophilized bulb 6 and the second lyophilized bulb 7 are always confined within the reagent compartment, preventing them from moving back towards the sample loading chamber or being sucked into the detection channel by the negative pressure source. This ensures that the lyophilized bulbs remain in a fixed position and their release is controllable throughout the transportation and detection process.

[0064] Preferably, the vertical part and horizontal part of the sample tube 1, the reagent tube 2 and the detection tube 3 are all cylindrical.

[0065] Furthermore, such as Figure 1 As shown, both the first and second mounting holes are stepped holes, with the larger diameter end of the hole. The inlet ends of reagent tube 2 and detection tube 3 are each provided with stepped shoulders that mate with the stepped holes. The first sealing ring 4 and the second sealing ring 5 are respectively placed on the axial stepped surfaces within the larger diameter ends of the first and second mounting holes; when reagent tube 2 and detection tube 3 are inserted into their corresponding mounting holes, their stepped shoulders axially press against the sealing rings, thereby achieving a seal.

[0066] Furthermore, the large-diameter sections of both the first and second mounting holes are machined with internal threads. The stepped shoulders of the reagent tube 2 and the detection tube 3 are each provided with corresponding external threads, so that the inlet section of the reagent tube 2 can be screwed into the first mounting hole, thereby forming a detachable sealed connection with the sample dispensing tube 1, and the inlet section of the detection tube 3 can be screwed into the second mounting hole, thereby forming a detachable sealed connection with the reagent tube 2.

[0067] Specifically, the endogenous activator includes one or more combinations of ellagic acid, kaolin, diatomaceous earth, and kaolin; the exogenous activator is calcium chloride; and the freeze-drying protectant mainly consists of trehalose, PVP (polyvinylpyrrolidone), bovine serum albumin, and Proclin 300.

[0068] Preferably, the endogenous activator is kaolin, and the concentration of kaolin before freeze-drying is 0.04 mg / mL. The concentration of calcium chloride before freeze-drying is 0.2 mol / L. The mass-volume concentrations of trehalose, PVP, bovine serum albumin, and Proclin 300 before freeze-drying are 10%, 6%, 3%, and 0.05%, respectively.

[0069] Preferably, the first freeze-dried pellet 6 and the second freeze-dried pellet 7 are obtained through the following process: the first solution and the second solution are frozen into pellets with a volume of 5 μL, and then freeze-dried according to the following process to obtain the first freeze-dried pellet 6 and the second freeze-dried pellet 7:

[0070] Pre-freeze at -43 to -47°C for 50 to 70 minutes;

[0071] Sublimation drying was carried out once at <10 Pa and -38 to -42 °C for 550 to 650 min.

[0072] Desorption and drying were carried out at <10 Pa and -37 to -33 °C for 50 to 70 minutes.

[0073] Desorption and drying were carried out under conditions of <10 Pa and -27 to -23 °C for 50 to 70 minutes;

[0074] Drying was carried out under conditions of <10 Pa and -17 to -13 °C for 50 to 70 minutes.

[0075] Drying was carried out under conditions of <10 Pa and -7 to -3 °C for 50 to 70 minutes.

[0076] Dry under conditions of <10 Pa and 3–7 °C for 50–70 min;

[0077] Desorption and drying were carried out at <10 Pa and 13-17℃ for 50-70 min;

[0078] Desorption and drying were carried out at <10 Pa and 23-27℃ for 300-400 min.

[0079] Preferably, the ultrasonic guided wave coagulation test reagent card is stored in a double-sealed vacuum aluminum foil bag, and has a shelf life of 2 years at room temperature (30°C).

[0080] like Figure 2 As shown, the lyophilized pellets obtained using the above-mentioned lyophilization protectant and through the above process are spherical, round, and smooth in appearance, with low moisture content and good solubility. This avoids the impact of high moisture content on the stability of the lyophilized pellet concentration and improves the stability of the lyophilized pellet performance. Generally speaking, if the lyophilized pellets have high moisture content, they are prone to moisture absorption, shrinkage, and collapse, affecting the test results of the reagents. However, this invention, by optimizing the lyophilization protectant and the lyophilization process, combined with a double-layer sealed packaging method using vacuum aluminum foil bags, ensures that when testing high and low value quality control samples of reagent cards stored at room temperature for 24 months, the repeatability CV is within 10% within the test value range of the quality control samples.

[0081] A second aspect of the present invention provides a coagulation detection method using the above-described ultrasonic guided wave coagulation detection reagent card.

[0082] The method of the present invention includes the following steps:

[0083] S1. Sodium citrate anticoagulated blood sample is injected into the sample loading chamber of the ultrasonic guided wave coagulation test reagent card. The sodium citrate anticoagulated blood sample flows into the reagent chamber and reacts with the first lyophilized bulb 6 and the second lyophilized bulb 7 to form a test sample. After the first lyophilized bulb 6 and the second lyophilized bulb 7 are fully mixed with the sodium citrate anticoagulated blood sample, the coagulation pathway is activated.

[0084] S2. Place the detection tube 3 under a static-dynamic composite magnetic field and connect the outlet of the detection channel to an external negative pressure source. Drive the test sample in the reagent chamber into the detection channel through the negative pressure applied by the negative pressure source until the sensitive element is immersed in the test sample.

[0085] In this step, the sensitive element in the detection channel undergoes ultrasonic-level vibration deformation under the combined action of a static magnetic field and a dynamic magnetic field.

[0086] In this step, since the detection tube 3 is made of transparent or semi-transparent material, a color sensor can be used to collect the color characteristics of the detection tube 3, and then the volume or liquid level of the detection sample in the detection tube 3 can be identified based on the color characteristics, thereby determining whether the sensitive element is submerged in the detection sample.

[0087] S3. Real-time detection of the resonant frequency or amplitude of the sensitive element, and construction of a curve showing the change of resonant frequency or amplitude over time;

[0088] In this step, the detected frequency includes, but is not limited to, the first, second, and third harmonics of the fundamental resonant frequency of the sensitive element. The fundamental resonant frequency of the sensitive element is related to its length.

[0089] S4. Generate a thromboelastography based on the curve of the resonant frequency or amplitude changing over time.

[0090] Specific embodiments of the present invention are as follows:

[0091] Example 1

[0092] In this embodiment, taking kaolin as an endogenous activator as an example, a method for preparing an ultrasonic guided wave coagulation test reagent card is provided, and the specific steps are as follows:

[0093] 1. Prepare a first solution and a second solution for freeze-drying based on the concentrations of kaolin, calcium chloride, and the freeze-drying protectant: the concentration of kaolin in the first solution is 0.04 mg / mL, and the concentration of calcium chloride in the second solution is 0.2 mol / L. The mass-volume concentrations of trehalose, PVP, bovine serum albumin, and Proclin 300 in the first and second solutions are 10%, 6%, 3%, and 0.05%, respectively.

[0094] 2. The first and second solutions were frozen into first and second spheres with a volume of 5 μL, respectively, using liquid nitrogen pre-freezing technology;

[0095] 3. Transfer the first and second pellets into a freeze dryer and freeze-dry them according to the following process to obtain the first and second freeze-dried pellets:

[0096] ① Pre-freeze at -45℃ for 300 minutes;

[0097] ② Sublime drying at 8 Pa and -40℃ for 600 min in one step;

[0098] ③ Dry under conditions of 8 Pa and -35℃ for 60 min;

[0099] ④ Dry under conditions of 8 Pa and -25℃ for 60 min;

[0100] ⑤ Dry under conditions of 8 Pa and -15℃ for 60 min;

[0101] ⑥ Dry under conditions of 8 Pa and -5℃ for 60 min;

[0102] ⑦ Dry under conditions of 8 Pa and 5 °C for 60 min;

[0103] ⑧ Dry under conditions of 8 Pa and 15 °C for 60 min;

[0104] ⑨ Dry under conditions of 8 Pa and 25 °C for 360 min.

[0105] 4. Place the first and second lyophilized bulbs into the reagent compartment of reagent tube 2. Screw the inlet end of reagent tube 2 into the first mounting hole of sample tube 1 using a threaded connection. After tightening, reagent tube 2 abuts against the first sealing ring 4, thus achieving a detachable and sealed connection with sample tube 1. After inserting the sensitive element into the detection channel from the inlet end of detection tube 3, screw the inlet end of detection tube 3 into the second mounting hole of the outlet end of reagent tube 2 using a threaded connection. After tightening, detection tube 3 abuts against the second sealing ring 5, thus achieving a detachable and sealed connection with reagent tube 2.

[0106] Example 2

[0107] This embodiment uses the ultrasonic guided wave coagulation test reagent card obtained in Example 1 to provide a coagulation test method.

[0108] In this embodiment, the whole blood sample to be tested first enters the reagent chamber through the sample compartment, the first and second lyophilized bulbs are dissolved, and finally flow into the detection channel for testing. The test is then performed using a micro-volume rapid ultrasonic guided wave coagulation elastography instrument (model: UTEG1.5 version, manufacturer: Taichuang Biomedical Technology (Huzhou) Co., Ltd.).

[0109] The detection method in this embodiment specifically includes the following steps:

[0110] 1.1 Collect at least 2 mL of venous whole blood using a sodium citrate anticoagulant blood collection tube;

[0111] 1.2 The collected samples should be stored at room temperature. Before testing, gently invert the blood collection tube at least 5 times to ensure that the sample is thoroughly mixed. Samples with obvious coagulation should not be used.

[0112] 1.3 Remove the ultrasonic guided wave coagulation test reagent card from the vacuum aluminum foil bag;

[0113] 1.4 Gently invert the blood collection tube containing venous whole blood at least 5 times to mix the sample, and use a pipette to add 27 μL of the sample into the sample dispensing chamber of the reagent card;

[0114] 1.5 Insert the reagent card into the detection slot of the instrument and use the micro-volume rapid ultrasonic guided wave coagulation elastography detector to complete the reagent card detection.

[0115] 1.6 The coagulation process takes about 5 to 10 minutes to complete. If the complete fibrinolysis process is to be observed, it needs to be extended to 30 minutes.

[0116] In this embodiment, the detection indicators include:

[0117] Blood clotting time R (min): 2–8; blood clot formation time K (min): 1–3; blood clotting rate MA (GPa): 2.5–3.2.

[0118] Example 3

[0119] This embodiment verifies the accuracy of the ultrasonic guided wave coagulation test reagent card obtained in Example 1.

[0120] 1. Select 5 fresh whole blood samples with different coagulation functions and their manufacturers:

[0121] ① Quality Control Level I (Beijing Adhauke)

[0122] 0 min < blood clotting time (R) ≤ 2.0 min;

[0123] 0 min < clot formation time (K) ≤ 5.0 min;

[0124] 60° < blood clotting rate (Angle) < 90°;

[0125] Blood clot strength (MA) ≤ 50.00 mm;

[0126] Main ingredient: Sodium citrate anticoagulated porcine plasma freeze-dried powder.

[0127] ② Quality Control II (Beijing Adhauke)

[0128] 2.0 min < blood clotting time (R);

[0129] 0 min < clot formation time (K) ≤ 5.0 min;

[0130] 60° < blood clotting rate (Angle) < 90°;

[0131] 50.00mm < clot strength (MA) ≤ 90.00mm;

[0132] Main ingredient: Sodium citrate anticoagulated porcine plasma freeze-dried powder.

[0133] The samples and quality control products I and II were measured using the micro-volume rapid ultrasonic guided wave coagulation test kit in Example 1 and the commercially available activated coagulation test kit, and the results were compared.

[0134] 2. The coagulation activation test kit (control reagent) includes: R1 is kaolin reagent, R2 is CaCl2 reagent, and sample cups, manufactured by Haemonetics, kit batch number HM01597. The control reagent is operated according to the corresponding procedure outlined in its kit.

[0135] 3. For the thromboelastography obtained above, quickly activate the coagulation reagent and test according to the procedure described above.

[0136] 4. The results of R, K, and MA determination for 5 samples and quality control I and II are shown in Table 3 below. From the table, it can be seen that the deviations between the test results of this reagent and the control reagent are all below ±10%, and the repeatability is less than 10%, indicating good accuracy.

[0137] Table 3. Accuracy testing of the micro-volume rapid ultrasonic guided wave coagulation test kit.

[0138]

[0139] Example 4

[0140] This embodiment verifies the stability of the micro-volume rapid ultrasonic guided wave coagulation test kit.

[0141] 1. Accelerate in a 37℃ constant temperature incubator for 0, 3, 6, and 9 days, and take quality control level 1 and quality control level 2. Use a micro-volume rapid ultrasonic guided wave coagulation test kit to test R, K, and MA.

[0142] 2. For the micro-volume rapid ultrasound guided wave coagulation test kit obtained above, the testing procedure is as described above.

[0143] 3. As can be seen from the table below, after 9 days of accelerated testing at 37℃, the repeatability of the test results is within 10%, indicating good accelerated stability.

[0144] ①37℃ Acceleration Day 0.

[0145]

[0146] ②37℃ Acceleration Day 3

[0147]

[0148]

[0149] ③Accelerated at 37℃ for 6 days.

[0150]

[0151] ④ Accelerated at 37℃ for 9 days.

[0152]

[0153]

[0154] Example 5

[0155] This embodiment demonstrates the long-term stability test of the micro-volume rapid ultrasonic guided wave coagulation test kit.

[0156] 1. Store at room temperature (30℃) for 0, 3, 6, 12, 18, and 24 months respectively. Take the same batch of quality control level 1 and quality control level 2 and test R, K, and MA using a micro-volume rapid ultrasound guided wave coagulation test kit.

[0157] 2. For the micro-volume rapid ultrasound guided wave coagulation test kit obtained above, the testing procedure is as described above.

[0158] 3. As can be seen from the table below, the repeatability test results of this reagent are all within 10% after being stored at room temperature for 24 months, indicating that this reagent has excellent long-term stability, which is superior to the shelf-life stability of existing reagents on the market.

[0159] ① Store at room temperature for 0 months.

[0160]

[0161]

[0162] ② Store at room temperature for 3 months.

[0163]

[0164] ③ Store at room temperature for 6 months

[0165]

[0166]

[0167] ④ Store at room temperature for 12 months.

[0168]

[0169] ⑥ Store at room temperature for 18 months.

[0170]

[0171]

[0172] ⑦ Store at room temperature for 24 months.

[0173]

[0174] The above specific embodiments are used to explain and illustrate the present invention, but not to limit the present invention. Any modifications and changes made to the present invention within the spirit and scope of the claims shall fall within the protection scope of the present invention.

[0175] The above description is only a preferred embodiment of the present invention. Therefore, all equivalent changes or modifications made to the structure, features and principles described in the claims of this patent application are included in the scope of this patent application.

Claims

1. An ultrasonic guided wave coagulation test reagent card comprising a sensitive element and a lyophilized bulb, comprising a sample dispensing chamber, a reagent chamber, and a detection channel connected in sequence, characterized in that: The reagent chamber contains a first lyophilized bulb (6) and a second lyophilized bulb (7). The first lyophilized bulb (6) and the second lyophilized bulb (7) are coated with an endogenous activator and an exogenous activator, respectively. Both the first lyophilized bulb (6) and the second lyophilized bulb (7) contain a lyophilization protectant. A sensitive element is placed in the detection channel. The sensitive element is made of a surface-treated magnetostrictive material. The ultrasonic guided wave coagulation test reagent card reflects the change in the viscosity of the test sample through the change in the resonant frequency or amplitude of the sensitive element.

2. The ultrasonic guided wave coagulation test reagent card according to claim 1, characterized in that: The magnetostrictive material includes permalloy, iron-cobalt-nickel alloy, pure nickel, and iron-based amorphous materials; the surface treatment is passivation treatment or UV-cured coating treatment.

3. The ultrasonic guided wave coagulation test reagent card according to claim 1 or 2, characterized in that: The sensitive element is an elongated structure, and the length direction of the elongated structure is parallel to the flow direction of the sample in the detection channel.

4. The ultrasonic guided wave coagulation test reagent card according to claim 3, characterized in that: The ultrasonic guided wave coagulation test reagent card is mainly composed of a sample dispensing tube (1), a reagent tube (2), a test tube (3), a first sealing ring (4), and a second sealing ring (5); The main body of the sample tube (1) is L-shaped, and a sample dispensing chamber is opened in the vertical part of the sample tube (1). A reagent chamber is opened through the reagent tube (2), and a detection channel is opened through the detection tube (3). The horizontal part of the sample tube (1) has a first mounting hole in the horizontal direction, and a first sealing ring (4) is arranged in the first mounting hole. One end of the reagent tube (2) is inserted into the first mounting hole and abuts against the first sealing ring (4), thereby detachably and sealingly connecting with the sample tube (1). The other end of the reagent tube (2) has a second mounting hole, and a second sealing ring (5) is arranged in the second mounting hole. One end of the detection tube (3) is inserted into the second mounting hole and abuts against the second sealing ring (5), thereby detachably and sealingly connecting with the reagent tube (2). The other end of the detection tube (3) is used to communicate with an external negative pressure source during the detection process. The detection tube (3) is made of transparent or semi-transparent material.

5. The ultrasonic guided wave coagulation test reagent card according to claim 4, characterized in that: The reagent chamber inside the reagent tube (2) includes a reagent compartment and a reagent compartment outlet section. The reagent compartment contains a first lyophilized bulb (6) and a second lyophilized bulb (7). The inner diameter of the reagent compartment outlet section is smaller than the inner diameter of the reagent compartment.

6. The ultrasonic guided wave coagulation test reagent card according to claim 1, characterized in that: The endogenous activator includes one or more combinations of ellagic acid, kaolin, diatomaceous earth, and kaolin; the exogenous activator is calcium chloride; the freeze-drying protectant mainly consists of trehalose, PVP, bovine serum albumin, and Proclin 300.

7. The ultrasonic guided wave coagulation test reagent card according to claim 5, characterized in that: The endogenous activator is kaolin, and the concentration of kaolin before freeze-drying is 0.04 mg / mL. The concentration of calcium chloride before freeze-drying is 0.2 mol / L. The mass-volume concentrations of trehalose, PVP, bovine serum albumin, and Proclin 300 before freeze-drying are 10%, 6%, 3%, and 0.05%, respectively. The ultrasonic guided wave coagulation test kit is stored in a double-sealed vacuum aluminum foil bag and has a shelf life of 2 years at room temperature.

8. The ultrasonic guided wave coagulation test reagent card according to claim 7, characterized in that: The first freeze-dried sphere (6) and the second freeze-dried sphere (7) are obtained through the following process: the first solution and the second solution are frozen into spheres with a volume of 5 μL, and then freeze-dried according to the following process to obtain the first freeze-dried sphere (6) and the second freeze-dried sphere (7): Pre-freeze at -43 to -47°C for 50 to 70 minutes; Sublimation drying was carried out once at <10 Pa and -38 to -42 °C for 550 to 650 min. Desorption and drying were carried out at <10 Pa and -37 to -33 °C for 50 to 70 minutes. Desorption and drying were carried out under conditions of <10 Pa and -27 to -23 °C for 50 to 70 minutes; Drying was carried out under conditions of <10 Pa and -17 to -13 °C for 50 to 70 minutes. Drying was carried out under conditions of <10 Pa and -7 to -3 °C for 50 to 70 minutes. Dry under conditions of <10 Pa and 3–7 °C for 50–70 min; Desorption and drying were carried out at <10 Pa and 13-17℃ for 50-70 min; Desorption and drying were carried out at <10 Pa and 23-27℃ for 300-400 min.

9. A method for coagulation detection using an ultrasonic guided wave coagulation test reagent card as described in any one of claims 1 to 8, characterized in that, Includes the following steps: S1. Sodium citrate anticoagulated blood sample is injected into the sample loading chamber of the ultrasonic guided coagulation test reagent card. The sodium citrate anticoagulated blood sample flows into the reagent chamber and reacts with the first lyophilized bulb (6) and the second lyophilized bulb (7) to form a test sample. S2. Place the detection tube (3) under a magnetic field and drive the detection sample into the detection channel through negative pressure. The sensitive element in the detection channel undergoes ultrasonic vibration deformation under the combined action of static and dynamic magnetic fields until the sensitive element is submerged in the detection sample. S3. Real-time detection of the resonant frequency or amplitude of the sensitive element, and construction of a curve showing the change of resonant frequency or amplitude over time; S4. Generate a thromboelastography based on the curve of the resonant frequency or amplitude changing over time.

10. The coagulation detection method according to claim 9, characterized in that: In step S3, the detected main frequency includes the first, second, and third harmonics of the fundamental resonant frequency of the sensitive element; The detection tube (3) is made of transparent or semi-transparent material. In step S2, a color sensor is used to collect the color characteristics of the detection tube (3) and then identify the volume or liquid level of the detection sample in the detection tube (3) to determine whether the sensitive element is submerged in the detection sample.

Images