A composite function body fluid collection tube

Abstract

This invention relates to the field of medical device technology and discloses a multifunctional body fluid collection tube. Addressing the problem that existing collection tubes cannot degrade cross-linked fibrin in body fluid samples, leading to high clot rates and cell retention, the body fluid collection tube of this invention has a dry-state multifunctional coating attached to its inner wall and bottom. The coating material includes an anticoagulant, plasminogen activator, D-(+)-trehalose dihydrate, D-mannitol, and phosphate buffer. During preparation, the anticoagulant, carbohydrate stabilizer, and buffer are mixed to form a base solution, then the plasminogen activator is added and mixed thoroughly. The mixture is then sprayed onto the inside of the tube and cured through a multi-step freeze-drying process. This invention utilizes a dual mechanism of anticoagulation and fibrinolysis to eliminate the physical retention of cells by fibrin, maintain cell suspension, and improve detection recovery rate. Specific excipient formulations combined with the freeze-drying process maintain the catalytic activity of biological components for long-term storage.

Description

Technical Field

[0001] This invention relates to the field of medical device technology, specifically to a multifunctional body fluid collection tube. Background Technology

[0002] Cell counting and differential diagnosis of body fluid specimens are important auxiliary means for clinical differentiation of transudates and exudates, and are also one of the routine laboratory tests for determining the nature of inflammation and suggesting malignant lesions. Body fluid collection tubes are specialized containers used for collecting and temporarily storing these body fluid specimens in vitro. The physicochemical properties of their internal pre-coated layer directly determine the integrity and monodispersity of cell morphology in the specimen, which is the physical basis for obtaining accurate cytological test results.

[0003] Currently, in routine clinical testing, vacuum blood collection tubes pre-filled with EDTA-2K or similar salts are commonly used to collect body fluid samples. In this procedure, EDTA-2K acts as a standard anticoagulant, dissolving rapidly after the body fluid is injected into the tube and chelating free calcium ions in the fluid through its molecular structure. Since the coagulation cascade depends on calcium ions as a cofactor, this chelation mechanism interrupts the conversion pathway from prothrombin to thrombin, thereby inhibiting subsequent coagulation reactions of the body fluid in an in vitro state.

[0004] However, existing anticoagulant collection tubes have a functional blind spot when processing pleural and peritoneal fluid specimens with high fibrin content. They can only passively prevent the formation of new clots, but cannot degrade existing cross-linked fibrin in the specimen. Conventional anticoagulants do not hydrolyze cross-linked fibrin networks that are already present in the patient's body fluids due to pathological factors or formed at the moment of sampling. These undissolved existing fibrin aggregate into clumps within the tube, encapsulating and physically trapping body fluid cells within the clot. This disrupts the cell suspension, preventing a large number of target cells from being absorbed by the detection instrument, ultimately resulting in a high clot rate in body fluid specimens and a significantly reduced recovery rate for cytological testing. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a composite-function body fluid collection tube that solves the problem that existing body fluid collection tubes cannot degrade the cross-linked fibrin in body fluid samples with high fibrin content, resulting in high sample agglutination rate and physical retention of cells by clots, thereby reducing the detection recovery rate and accuracy.

[0006] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a multifunctional body fluid collection tube, which adopts the following technical solution: A multifunctional body fluid collection tube includes a tube body, the inner wall and bottom of which are coated with a dry multifunctional coating. The multifunctional coating is made of raw materials containing the following components: anticoagulant, plasminogen activator, D-(+)-trehalose dihydrate, D-mannitol and phosphate buffer. The effective amounts of the raw material components contained in the tube body, based on a preset collection volume of 1 mL of body fluid, are: anticoagulant 1.5–2.5 mg, plasminogen activator 50–500 IU, D-(+)-trehalose dihydrate 0.5–2.0 mg, and D-mannitol 0.2–1.0 mg.

[0007] By adopting the above technical solution, the technical effects of reducing the incidence of clots in body fluid samples and improving cell recovery rate were achieved. The specific biochemical reaction mechanism is as follows: In the first step, after the body fluid is injected into the tube, the coating dissolves instantly. The anticoagulant enters the liquid phase, and its molecular structure undergoes a complexation reaction with free calcium ions in the body fluid. The coagulation cascade reaction is highly dependent on free calcium ions as a cofactor. This complexation depletes calcium ions, blocks the conversion of prothrombin to thrombin, thereby inhibiting the conversion of fibrinogen to fibrin and blocking the pathway for new clot formation.

[0008] In the second step, plasminogen activator works simultaneously. As a specific serine protease, it catalyzes the conversion of existing plasminogen in the body fluid sample into active plasmin. The activated plasmin then hydrolyzes cross-linked fibrin peptide bonds, degrading fibrin into soluble D-dimers and fibrin degradation products, eliminating the clot structure that existed before the body fluid left the body. Anticoagulation and fibrinolysis occur simultaneously, completely breaking the physical encapsulation of cells by the clot.

[0009] In the third step, D-(+)-trehalose dihydrate within the coating system forms stable hydrogen bonds with polar amino acid residues on the surface of plasminogen activator in a dehydrated state, replacing the hydration of water molecules to maintain the protein's folded conformation. D-mannitol, as a crystalline framework support, restricts the molecular movement of polypeptide chains in the amorphous phase. Both work synergistically to maintain the catalytic activity of biological enzymes in the solid-phase state.

[0010] Preferably, based on a pre-set 1 mL body fluid collection volume in the tube, the optimal effective amounts of the raw material components contained in the tube are: 2.0 mg anticoagulant, 200 IU plasminogen activator, 1.0 mg D-(+)-trehalose dihydrate, and 0.5 mg D-mannitol. The anticoagulant is either ethylenediaminetetraacetic acid dipotassium dihydrate or ethylenediaminetetraacetic acid tripotassium dihydrate; the plasminogen activator is either recombinant tissue-type plasminogen activator or urokinase. The phosphate buffer used in preparing the composite functional coating has a molar concentration of 0.05–0.15 mol / L and a pH of 7.0–7.4.

[0011] By employing the above technical solution, the parameters of the phosphate buffer solution maintain an isotonic and acid-base balanced environment in the reconstitution system that is similar to that of human body fluids. This avoids hypertonic conditions caused by excessively high local salt concentrations, prevents osmotic rupture of blood cells, and ensures the physical integrity of isolated cells.

[0012] Preferably, the composite functional coating has a porous skeleton structure, and the residual moisture content of the composite functional coating is controlled to be 1.0% to 3.0% by mass.

[0013] By employing the above technical solution, the porous framework structure increases the specific surface area of ​​the coating, providing a physical channel for the penetration of liquid phase molecules and improving the dissolution rate. The controlled moisture range within a specific region avoids irreversible unwinding of the protein's spatial structure caused by complete dehydration stress, while also limiting free water molecule-mediated chemical degradation reactions.

[0014] Secondly, the present invention provides a coating preparation method for a composite functional body fluid collection tube, employing the following technical solution: A method for preparing a coating for a composite functional body fluid collection tube includes the following steps: S100, preparation of composite mother liquor: Under aseptic conditions, anticoagulant, D-(+)-trehalose dihydrate and D-mannitol are dissolved in phosphate buffer to prepare basic protective solution; then plasminogen activator is added and stirred under low shear force to prepare composite functional mother liquor; S200, Inner wall spraying: Using atomized micro-spraying equipment, the composite functional mother liquor obtained in step S100 is quantitatively sprayed onto the lower middle part of the inner wall of the pipe and the bottom area of ​​the pipe. S300, freeze-drying curing: The coated tube is placed in a freeze dryer and a step freeze-drying process including pre-freezing, first sublimation drying and second desorption drying is performed to obtain a dry composite functional coating in situ inside the tube.

[0015] By employing the above technical solution, the problem of thermal denaturation and inactivation of enzymes and proteins caused by conventional heating and drying is overcome. Step S100 involves adding protein components susceptible to mechanical damage later, maintaining their structural integrity. Step S200 alters the liquid phase distribution, preventing solute aggregation and surface hardening. Step S300 induces low-temperature phase transition crystallization, followed by direct sublimation and dehydration of solid ice in a vacuum environment, generating a porous three-dimensional network framework in situ, thus achieving stable solid-phase retention of the active ingredients.

[0016] Preferably, in step S100, the specific parameters for preparing the basic protective solution are: temperature controlled at 20–25°C, stirring speed at 300–500 r / min, and time at 15–30 minutes; the specific parameters after adding the plasminogen activator are: temperature controlled at 2–8°C and kept in the dark, stirring speed at 100–200 r / min, and time at 10–20 minutes. Step S200 is implemented as follows: using an ultrasonic atomizing micro-spraying device, setting the atomized droplet size range during spraying to 20–50 μm; based on a preset collection volume of 3 mL of body fluid, the quantitative spraying loading volume of the single-tube composite functional mother liquor is 50–150 L.

[0017] By employing the above technical solutions, the combination of a low-temperature, light-protected environment and low shear force conditions reduces the degradation of protein molecules caused by external energy. The limited micron-sized atomized droplets improve the uniformity of the coating distribution on the tube wall, ensuring consistent dissolution rates in all areas during reconstitution and preventing drastic local fluctuations in biochemical reaction concentrations.

[0018] Preferably, the specific process parameters for step S300 are as follows: Pre-freezing stage: The plate temperature is linearly reduced to -45 to -40°C at a cooling rate of 1.0–2.5°C / min, and held at this temperature for 120–180 minutes; First sublimation drying stage: The ambient absolute pressure is reduced to 10–20 Pa, while the plate temperature is increased to -15 to -10°C at a heating rate of 0.5–1.0°C / min, and held at this temperature for 300–480 minutes; Secondary desorption drying stage: The ambient absolute pressure is maintained at 10–20 Pa, and the plate temperature is increased to 20–25°C at a rate of 1.0–1.5°C / min, and held at this temperature for 180–240 minutes. Following step S300, the following post-processing steps are also included: S400, in a nitrogen-protected production line with a purity of 99.9%, establishes a corresponding internal negative pressure environment according to the preset body fluid collection volume of the tube, and presses the tube cap and sealing component onto the top of the tube to complete the airtight sealing. S500 sterilizes the finished tubes with cobalt-60 radiation by penetrating irradiation after the airtight sealing is completed, and controls the absorbed dose range of 8 to 15 kGy during the irradiation process.

[0019] By employing the above technical solution, the stepped temperature control curve ensures complete crystallization of the liquid system and the smooth removal of free and bound water, preventing solution boiling and structural collapse during the phase transition process. Subsequent nitrogen-filled encapsulation meets the requirements of clinical vacuum blood collection. The limited dose of radiation irradiation achieves sterility assurance while avoiding excessive radiation energy from severing the protein and polypeptide chain backbone, ensuring that the collection tube possesses both sterility and biochemical catalytic activity. This invention provides a multifunctional body fluid collection tube. It has the following beneficial effects: 1. This invention reduces the clot formation rate and improves cell recovery rate in body fluid samples. A composite functional coating composed of an anticoagulant and a plasminogen activator is pre-placed inside the tube. The anticoagulant inhibits new clot formation by chelating free calcium ions and blocking the coagulation pathway. The plasminogen activator catalyzes the conversion of plasminogen in the body fluid into plasmin, hydrolyzing existing cross-linked fibrin. This dual mechanism eliminates the physical trapping of body fluid cells by fibrin, maintains the monodisperse suspension of cells, and improves the detection rate of body fluid cells.

[0020] 2. This invention maintains the catalytic activity of bioactive components during long-term storage and sterilization. The composite functional coating uses D-(+)-trehalose dihydrate and D-mannitol as a protective matrix. Trehalose forms hydrogen bonds with the polar groups of plasminogen activator in a solid-phase dehydrated state to maintain the spatial folding conformation of its polypeptide chain, while mannitol provides crystalline scaffold support to restrict protein molecule movement. This formulation, combined with freeze-drying technology, avoids protein unwinding and denaturation caused by radiation irradiation and long-term storage environments, thus extending the product's effective shelf life.

[0021] 3. This invention improves the resolution rate of the coating while maintaining the structural integrity of body fluid cells. The preparation process employs ultrasonic atomization spraying combined with stepwise freeze-drying, enabling the composite functional coating to form a porous framework structure on the tube wall, increasing the specific surface area. Liquid molecules can then rapidly penetrate and dissolve after injection. A specific concentration of phosphate buffer solution is used as the solvent base within the composite coating system, maintaining the isotonicity and acid-base balance of the system after resolution, preventing osmotic rupture of body fluid cells due to localized hyperosmolarity. Attached Figure Description

[0022] Figure 1 This is a perspective view of the present invention.

[0023] Among them, 1. Pipe body; 2. Pipe cap. Detailed Implementation

[0024] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and 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.

[0025] Please see the appendix Figure 1 This invention provides a composite function body fluid collection tube, including a tube body 1, a tube cap 2 threadedly connected to the upper end of the tube body 1, and a composite function coating provided on the bottom and middle of the inner surface of the tube body 1. The main raw materials and reagents used in the following examples and comparative examples have the following sources and specifications. Reagents not specifically mentioned are all commercially available analytical grade or higher grade products.

[0026] Among them, the anticoagulant components, ethylenediaminetetraacetic acid dipotassium dihydrate (CAS No. 25102-12-9) and ethylenediaminetetraacetic acid tripotassium dihydrate (CAS No. 65501-24-8), are commercially available analytical grade solid powder reagents. The immediate fibrinolytic component recombinant tissue plasminogen activator (CAS No. 105857-23-6), urokinase (CAS No. 9039-53-6), and plasminogen as a substrate reserve (CAS No. 9001-91-6) are all commercially available biotechnology-grade high-purity lyophilized powder reagents. The D-(+)-trehalose dihydrate (CAS No. 6138-23-4) and D-mannitol (CAS No. 69-65-8) used in the freeze-drying protective matrix are both commercially available biotechnology-grade solid powder reagents. The phosphate buffer and other basic solvents used in the formulation to maintain isotonicity and acid-base balance are all conventionally known substances in the field and can be obtained directly through conventional commercial channels. They do not have specific molecular stereostructures or preparation process limitations, so unnecessary descriptions of their structures and preparation parameters will not be provided here.

[0027] Preparation Example 1: This preparation example provides a composite functional stock solution for anticoagulation and immediate fibrinolysis composite coatings, comprising the following steps: Under aseptic conditions controlled at room temperature (25°C), accurately weigh 6.0 g of ethylenediaminetetraacetic acid dipotassium dihydrate, 3.0 g of D-(+)-trehalose dihydrate, and 1.5 g of D-mannitol, and add them sequentially to 100 mL of 0.1 mol / L phosphate buffer with a pH of 7.2. Stir continuously at 400 r / min for 20 minutes using a magnetic stirrer until the solid powder is completely dissolved to obtain a basic protective solution. Subsequently, lyophilized powder or stock solution of recombinant tissue plasminogen activator with a total activity of 600,000 IU is slowly added to the basic protective solution. Under light-protected conditions at 4°C, the mixture is stirred continuously at 150 r / min with low shear force for 15 minutes to obtain a homogeneous composite functional stock solution. When the mother liquor is sprayed in a single tube at a concentration of 100L and used to collect 3mL of body fluid, the final equivalent concentration of the anticoagulant in the tube is 2.0mg / mL, and the final equivalent concentration of the plasminogen activator is 200IU / mL.

[0028] Preparation Example 2: This preparation example provides a composite functional stock solution for an anticoagulant and instant fibrinolytic composite coating, comprising the following steps: Under aseptic conditions controlled at room temperature (20°C), accurately weigh 4.5 g of ethylenediaminetetraacetic acid dipotassium dihydrate, 1.5 g of D-(+)-trehalose dihydrate, and 0.6 g of D-mannitol, and add them sequentially to 100 mL of 0.05 mol / L phosphate buffer with a pH of 7.0. Stir continuously at 300 r / min for 15 minutes using a magnetic stirrer until the solid powder is completely dissolved to obtain a basic protective solution. Subsequently, lyophilized powder or stock solution of recombinant tissue plasminogen activator with a total activity of 150,000 IU is slowly added to the basic protective solution. Under light-protected conditions at 2°C, the mixture is stirred continuously at 100 r / min with low shear force for 10 minutes to obtain a homogeneous composite functional stock solution. When the mother liquor is sprayed in a single tube at a concentration of 100L and used to collect 3mL of body fluid, the final equivalent concentration of the anticoagulant in the tube is 1.5mg / mL, and the final equivalent concentration of the plasminogen activator is 50IU / mL.

[0029] Preparation Example 3: This preparation example provides a composite functional stock solution for an anticoagulant and instant fibrinolytic composite coating, comprising the following steps: Under aseptic conditions controlled at room temperature (25°C), accurately weigh 7.5 g of ethylenediaminetetraacetic acid dipotassium dihydrate, 6.0 g of D-(+)-trehalose dihydrate, and 3.0 g of D-mannitol, and add them sequentially to 100 mL of 0.15 mol / L phosphate buffer with a pH of 7.4. Stir continuously at 500 r / min for 30 minutes using a magnetic stirrer until the solid powder is completely dissolved to obtain a basic protective solution. Subsequently, lyophilized powder or stock solution of recombinant tissue plasminogen activator with a total activity of 1,500,000 IU is slowly added to the basic protective solution. Under light-protected conditions at 8°C, the mixture is stirred continuously at 200 r / min with low shear force for 20 minutes to obtain a homogeneous composite functional stock solution. When the mother liquor is sprayed in a single tube at a concentration of 100L and used to collect 3mL of body fluid, the final equivalent concentration of the anticoagulant in the tube is 2.5mg / mL, and the final equivalent concentration of the plasminogen activator is 500IU / mL.

[0030] Example 1: This embodiment provides a method for preparing an anticoagulant + instant fibrinolytic composite functional body fluid collection tube, including the following steps: S100, prepare the composite functional mother liquor according to the method described in Preparation Example 1; S200 uses an ultrasonic atomizing micro-spraying device to quantitatively and uniformly adhere the composite functional mother liquor obtained in Preparation Example 1 to the lower middle part and bottom area of ​​the inner wall of a 5mL vacuum blood collection tube 1. The spraying loading volume of the composite functional mother liquor per tube is strictly controlled at 100L, and the atomized droplet particle size is set to 35μm during the spraying operation.

[0031] In S300, the coated tube 1 is transferred to the vacuum chamber of the freeze dryer for a stepped freeze-drying process, including pre-freezing, primary sublimation drying, and secondary desorption drying. During the pre-freezing stage, the plate temperature is linearly reduced to -42°C at a rate of 1.5°C / min and held constant for 150 minutes, allowing the composite functional mother liquor system inside tube 1 to undergo complete crystallization phase change. In the primary drying stage, the vacuum pump is activated to reduce the absolute pressure in the drying chamber to 15 Pa, while the plate temperature is slowly increased to -12°C at a rate of 0.8°C / min and held constant for 390 minutes. In the secondary drying stage, while maintaining a vacuum of 15 Pa, the plate temperature is increased to 22°C at a rate of 1.2°C / min and held constant for 210 minutes, forming a dry composite functional coating in situ on the inner wall and bottom of tube 1.

[0032] In the S400, the vacuum capping production line is connected to a nitrogen gas supply line with a purity of 99.9%. The internal negative pressure environment is established according to the preset 3mL body fluid collection volume of tube 1. The pre-assembled tube cap 2 and sealing component are pressed together at the top opening of tube 1 to complete the airtight sealing.

[0033] S500 involves sterilizing the finished tube body 1 with mechanical seal by cobalt-60 irradiation, with the absorbed dose during irradiation limited to 10 kGy.

[0034] Example 2: This embodiment provides a method for preparing an anticoagulant + instant fibrinolytic composite functional body fluid collection tube. This embodiment verifies the method using the lower limits of the component ratio parameter range and the lower limits of the freeze-drying process parameters, and includes the following steps: S100, prepare the composite functional mother liquor according to the method described in Preparation Example 2; S200, using an ultrasonic atomizing micro-spraying device, quantitatively and uniformly adheres the composite functional mother liquor obtained in Preparation Example 2 to the lower middle part and bottom area of ​​the inner wall of a 5mL vacuum blood collection tube 1. The spraying loading volume of the composite functional mother liquor per tube is strictly controlled at 100L, and the atomized droplet particle size is set to 20μm during the spraying operation.

[0035] In S300, the coated tube 1 is transferred to the vacuum chamber of the freeze dryer for a stepped freeze-drying process, including pre-freezing, primary sublimation drying, and secondary desorption drying. During the pre-freezing stage, the plate temperature is linearly reduced to -40°C at a rate of 1.0°C / min and held constant for 120 minutes, allowing the composite functional mother liquor system inside tube 1 to undergo complete crystallization phase change. In the primary drying stage, the vacuum pump is activated to reduce the absolute pressure in the drying chamber to 10 Pa, while the plate temperature is slowly increased to -10°C at a rate of 0.5°C / min and held constant for 300 minutes. In the secondary drying stage, while maintaining a vacuum of 10 Pa, the plate temperature is increased to 20°C at a rate of 1.0°C / min and held constant for 180 minutes, forming a dry composite functional coating in situ on the inner wall and bottom of tube 1.

[0036] In the S400, the vacuum capping production line is connected to a nitrogen gas supply line with a purity of 99.9%. The internal negative pressure environment is established according to the preset 3mL body fluid collection volume of tube 1. The pre-assembled tube cap 2 and sealing component are pressed together at the top opening of tube 1 to complete the airtight sealing.

[0037] S500 involves sterilizing the finished tube body 1 with mechanical seal by cobalt-60 irradiation, with the absorbed dose during irradiation limited to 8 kGy.

[0038] Example 3: This embodiment provides a method for preparing an anticoagulant + instant fibrinolytic composite functional body fluid collection tube. This embodiment verifies the method using the upper limit of the component ratio parameter range and the upper limit of the freeze-drying process parameters, and includes the following steps: S100, prepare the composite functional mother liquor according to the method described in Preparation Example 3; S200, using an ultrasonic atomizing micro-spraying device, quantitatively and uniformly adheres the composite functional mother liquor obtained in Preparation Example 3 to the lower middle part and bottom area of ​​the inner wall of a 5mL vacuum blood collection tube 1. The spraying loading volume of the composite functional mother liquor per tube is strictly controlled at 100L, and the atomized droplet particle size is set to 50μm during the spraying operation.

[0039] In S300, the coated tube 1 is transferred to the vacuum chamber of the freeze dryer for a stepped freeze-drying process, including pre-freezing, primary sublimation drying, and secondary desorption drying. During the pre-freezing stage, the plate temperature is linearly reduced to -45°C at a rate of 2.5°C / min and held constant for 180 minutes, allowing the composite functional mother liquor system inside tube 1 to undergo complete crystallization phase change. In the primary drying stage, the vacuum pump is activated to reduce the absolute pressure in the drying chamber to 20 Pa, while the plate temperature is slowly increased to -15°C at a rate of 1.0°C / min and held constant for 480 minutes. In the secondary drying stage, while maintaining a vacuum of 20 Pa, the plate temperature is increased to 25°C at a rate of 1.5°C / min and held constant for 240 minutes, forming a dry composite functional coating in situ on the inner wall and bottom of tube 1.

[0040] In the S400, the vacuum capping production line is connected to a nitrogen gas supply line with a purity of 99.9%. The internal negative pressure environment is established according to the preset 3mL body fluid collection volume of tube 1. The pre-assembled tube cap 2 and sealing component are pressed together at the top opening of tube 1 to complete the airtight sealing.

[0041] S500 involves sterilizing the finished tube body 1 with mechanical seal by cobalt-60 irradiation, with the absorbed dose during irradiation limited to 15 kGy.

[0042] Example 4: This embodiment provides a method for preparing an anticoagulant + instant fibrinolytic composite functional body fluid collection tube. This embodiment uses equivalent substitution of active substances for verification, including the following steps: S100, under aseptic conditions controlled at 25℃, accurately weighed 6.0g of ethylenediaminetetraacetic acid tripotassium dihydrate, 3.0g of D-(+)-trehalose dihydrate, and 1.5g of D-mannitol, and added them sequentially to 100mL of 0.1mol / L phosphate buffer with a pH of 7.2. The mixture was stirred continuously at 400 rpm for 20 minutes using a magnetic stirrer until the solid powder was completely dissolved, thus preparing the basic protective solution. Subsequently, 900,000 IU of urokinase lyophilized powder was slowly added to the basic protective solution, and stirred continuously at 150 rpm for 15 minutes under low shear force conditions at 4℃ in the dark, to obtain a homogeneous equivalent multifunctional stock solution.

[0043] In step S200, an ultrasonic atomizing micro-spraying device is used to quantitatively and uniformly adhere the equivalent composite functional mother liquor obtained in step S100 of this embodiment to the lower middle part of the inner wall and the bottom area of ​​the tube body 1 of a 5mL vacuum blood collection tube. The spraying loading volume of the equivalent composite functional mother liquor per tube is strictly controlled at 100L, and the atomized droplet particle size is set to 35μm during the spraying operation.

[0044] In S300, the coated tube 1 is transferred to the vacuum chamber of the freeze dryer for a stepped freeze-drying process, including pre-freezing, primary sublimation drying, and secondary desorption drying. During the pre-freezing stage, the plate temperature is linearly reduced to -42°C at a rate of 1.5°C / min and held at this temperature for 150 minutes. In the primary drying stage, the vacuum pump is activated to reduce the absolute pressure in the drying chamber to 15 Pa, while the plate temperature is slowly increased to -12°C at a rate of 0.8°C / min and held at this temperature for 390 minutes. In the secondary drying stage, while maintaining a vacuum of 15 Pa, the plate temperature is increased to 22°C at a rate of 1.2°C / min and held at this temperature for 210 minutes, resulting in a dry composite functional coating forming in situ on the inner wall and bottom of tube 1.

[0045] In the S400, the vacuum capping production line is connected to a nitrogen gas supply line with a purity of 99.9%. The internal negative pressure environment is established according to the preset 3mL body fluid collection volume of tube 1. The pre-assembled tube cap 2 and sealing component are pressed together at the top opening of tube 1 to complete the airtight sealing.

[0046] S500 involves sterilizing the finished tube body 1 with mechanical seal by cobalt-60 irradiation, with the absorbed dose during irradiation limited to 10 kGy.

[0047] Comparative Example 1: Compared with Example 1, the difference is that no recombinant tissue plasminogen activator was added during the preparation of the compound functional mother liquor, and all other aspects are the same.

[0048] Comparative Example 2: Compared with Example 1, the difference is that ethylenediaminetetraacetic acid dipotassium dihydrate was not added during the preparation of the composite functional mother liquor, while all other aspects are the same.

[0049] Comparative Example 3: Compared with Example 1, the difference is that D-(+)-trehalose dihydrate and D-mannitol were not added during the preparation of the composite functional mother liquor, and only 0.1 mol / L phosphate buffer was used as the basic protective solution, while the rest were the same.

[0050] Comparative Example 4: Compared with Example 1, the difference is that in the preparation of the composite functional mother liquor, the amount of ethylenediaminetetraacetic acid dipotassium dihydrate was changed to 15.0g (that is, the final concentration of anticoagulant in the target acquisition system after single-tube spraying exceeded the standard to 5.0mg / mL), and all other aspects were the same.

[0051] Comparative Example 5: Compared with Example 1, the difference is that the multi-step freeze-drying procedure in step S300 was omitted, and the coated tube was directly placed in a constant temperature vacuum drying oven at 37°C for conventional heating, drying and curing. All other aspects are the same.

[0052] Test Example 1: Basic Physicochemical Indicators and Protein Conformation Maintenance Performance Test of Composite Functional Body Fluid Collection Tube This test case aims to verify the coating moisture control level, fluid resolution kinetics characteristics, and activity retention status of key bioactive components in the products prepared by the examples and some comparative examples in the specification after curing and sterilization processes.

[0053] Take the collection tubes from Examples 1 to 4 and Comparative Examples 3 and 5, and scrape off the dry coating from the inner wall and bottom of the tube in an environment with a relative humidity of less than 20%. Perform moisture titration analysis using a Karl Fischer coulometric moisture analyzer and record the residual moisture mass fraction. Inject 3 mL of room temperature purified water into the sampling tube, fix it on a gyroscope, set the oscillation frequency to 60 times / minute and the amplitude to 20 mm, and record the time from water contact with the coating until the coating particles are no longer visible and the solution becomes transparent. Extract the reconstituted liquid sample, and determine the catalytic activity of serine protease in the system using the S-2251 chromogenic substrate method. Add plasminogen and chromogenic substrate to the sample, incubate at 37°C, and read the absorbance change rate at 405 nm using a microplate reader. Calculate the relative enzyme activity retention percentage after reconstitution using the initial absorbance change rate of the same batch of un-lyophilized and un-irradiated liquid multifunctional mother liquor as a benchmark. Table 1. Physicochemical properties and enzyme activity maintenance test results of the examples and comparative products. In the example group, the moisture content of the coating was between 1.0% and 3.0%, and the resolution time was less than 5 seconds. Atomized spraying applied the mother liquor as droplets of 20 to 50 μm to the pipe wall. Combined with ice crystal nucleation during the pre-freezing process and ice crystal sublimation during the primary drying stage, a porous framework structure was formed on the pipe wall, providing fluid channels for liquid penetration and preventing solute surface hardening. In Comparative Example 5, conventional heating drying resulted in insufficient moisture removal, causing the coating structure to collapse and prolonging the resolution time.

[0054] After dehydration and irradiation, the enzyme activity retention rate of the example group remained above 93%. Trehalose, as a non-reducing disaccharide in the formulation, forms hydrogen bonds with polar groups on the protease surface by replacing water molecules in its molecular structure during drying to remove the protein hydration layer, thus maintaining the protein's spatial conformation. Mannitol provides skeletal rigidity, restricting polypeptide chain movement. In Comparative Example 3, without added carbohydrate stabilizers, the enzyme molecules underwent structural unwinding due to dehydration stress and radiation, resulting in an enzyme activity reduction to 42.6%. Comparative Example 5 showed denaturation and inactivation due to heat. The combination of coating composition and a multi-step freeze-drying process achieved the stability of the composite additive within the tube.

[0055] Test Example 2: Clot Degradation and Cell Recovery Performance Test of Composite Functional Body Fluid Collection Tube This test case aims to verify the synergistic effect of the products prepared by the examples and some comparative examples in the specification on anticoagulation and fibrinolysis when processing body fluid samples containing cross-linked fibrin, and their impact on the cell recovery rate in the samples.

[0056] Prepare simulated body fluid samples containing cross-linked fibrin and a fixed leukocyte concentration. Inject 3 mL of the simulated body fluid sample into the collection tubes of Examples 1 to 4 and Comparative Examples 1, 2, and 4, respectively. Mix by inverting 5 times and let stand at room temperature for 30 minutes. Filter the liquid through a 100-mesh sieve, collect and weigh the clot trapped by the sieve. Take the filtered liquid and determine the leukocyte concentration using an automated hematology analyzer to calculate the leukocyte recovery rate. Centrifuge a portion of the filtered liquid, separate the supernatant, and determine the D-dimer concentration using immunoturbidimetry.

[0057] Table 2. Sample processing performance test results of the sample collection tubes in the examples and comparative examples. Test data indicate that the anticoagulant component and plasminogen activator have a synergistic effect. In Comparative Example 1, no recombinant tissue plasminogen activator was added; cross-linked fibrin in the sample was not degraded, resulting in cell retention in the clot. The filter intercepted a high amount of clot, leading to a low white blood cell recovery rate. In Comparative Example 2, no anticoagulant was added; free calcium ions mediated the coagulation reaction, generating fibrin. The degradation rate of the single fibrinolytic component was lower than the clot formation rate, resulting in clot residue in the tube. In Comparative Example 4, the anticoagulant concentration was too high, creating a hypertonic environment in the tube, causing some blood cells to rupture, resulting in a low cell recovery rate.

[0058] In the example group, dipotassium ethylenediaminetetraacetate (EDTA) complexes with calcium ions, blocking the coagulation pathway and inhibiting the formation of new clots; recombinant tissue-type plasminogen activator catalyzes the conversion of plasminogen to plasmin, hydrolyzing fibrin. Increased D-dimer concentration corresponds to the fibrin degradation process. The dual mechanism of anticoagulation and fibrinolysis eliminates clot encapsulation of cells, maintains leukocyte suspension, and improves cell recovery rate.

[0059] Test Example 3: Accelerated Aging and Long-Term Storage Stability Test of Composite Functional Body Fluid Collection Tube This test example aims to verify the effect of the freeze-dried protective matrix component of the product prepared according to the embodiments of the specification on maintaining the spatial conformation and catalytic activity of the contained plasminogen activator under high temperature and high humidity environmental stress.

[0060] The collection tubes from Examples 1 to 4 and Comparative Example 3 were placed in a constant temperature and humidity incubator at 37°C and 75% relative humidity. Samples were collected on days 0, 15, 30, 60, and 90. 3 mL of purified water was added to each collected tube to reconstitute the sample. The catalytic activity of serine protease in the liquid phase system was determined using the S-2251 chromogenic substrate method. The relative enzyme activity retention rate at each time point was calculated based on the absorbance change rate measured on day 0.

[0061] Table 3. Results of accelerated aging test enzyme activity retention rate of the sample tubes in the examples and comparative examples. The freeze-drying protective matrix system maintained the structural stability of plasminogen activator under storage conditions. Under accelerated aging conditions at 37°C, Comparative Example 3, without the addition of D-(+)-trehalose dihydrate and D-mannitol, exposed the active component to the heat environment, resulting in unwinding and denaturation of the protein's secondary and tertiary structures. On day 30, the enzyme activity decreased to 18.6%.

[0062] In the example group, the enzyme activity retention rate was above 80% after 90 days of aging. D-(+)-trehalose dihydrate exists in an amorphous glassy state within the coating. The hydroxyl groups in its molecular structure replace water molecules, forming hydrogen bonds with polar amino acid residues on the surface of the plasminogen activator, maintaining the folded conformation of the polypeptide chain. D-mannitol acts as a skeletal support, reducing the degree of freedom of polypeptide chain movement and inhibiting protein aggregation and precipitation. The anticoagulant exhibits high thermal stability. The compatibility of the excipients inhibits the degradation and inactivation of the biological enzyme preparation, meeting the requirements for long-term storage of blood collection tubes.

[0063] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A composite function body fluid collection tube, comprising a tube body (1), the inner wall and the bottom of the tube body (1) are attached with a dry composite function coating; the composite function coating is made of raw materials containing the following components: anticoagulant, plasminogen activator, D- (+) -trehalose dihydrate, D-mannitol and phosphate buffer; wherein, The tube (1) is designed to collect 1 mL of body fluid. The effective amount of the raw material components contained in the tube (1) is as follows: 1.5-2.5 mg of the anticoagulant, 50-500 IU of the plasminogen activator, 0.5-2.0 mg of D-(+)-trehalose dihydrate, and 0.2-1.0 mg of D-mannitol.

2. The composite functional bodily fluid collection tube of claim 1, wherein, According to the preset 1mL body fluid collection meter of the tube (1), the preferred effective amount of the raw material components contained in the tube (1) is: 2.0mg of the anticoagulant, 200IU of the plasminogen activator, 1.0mg of D-(+)-trehalose dihydrate, and 0.5mg of D-mannitol.

3. The composite functional bodily fluid collection tube of claim 1, wherein, The anticoagulant is ethylenediaminetetraacetic acid dipotassium dihydrate or ethylenediaminetetraacetic acid tripotassium dihydrate; the plasminogen activator is recombinant tissue plasminogen activator or urokinase.

4. The composite functional bodily fluid collection tube of claim 1, wherein, The phosphate buffer solution used in preparing the composite functional coating has a molar concentration of 0.05–0.15 mol / L and a pH value of 7.0–7.

4.

5. The composite functional bodily fluid collection tube of claim 1, wherein, The composite functional coating has a porous skeleton structure, and the residual moisture content of the composite functional coating is controlled to be 1.0% to 3.0% by mass.

6. A method for preparing a coating of a composite functional body fluid collection tube, applied to the composite functional body fluid collection tube according to any one of claims 1 to 5, characterized in that, Includes the following steps: S100, preparation of composite mother liquor: Under aseptic conditions, anticoagulant, D-(+)-trehalose dihydrate and D-mannitol are dissolved in phosphate buffer to prepare basic protective solution; then plasminogen activator is added and stirred under low shear force to prepare composite functional mother liquor; S200, Inner wall spraying: Using atomized micro-spraying equipment, the composite functional mother liquor obtained in step S100 is quantitatively sprayed onto the lower middle part of the inner wall of the pipe and the bottom area of ​​the pipe. S300, freeze-drying curing: The coated tube is placed in a freeze dryer and a step freeze-drying process including pre-freezing, first sublimation drying and second desorption drying is performed to obtain the composite functional coating in situ inside the tube.

7. The coating production method according to claim 6, characterized in that In step S100, the specific parameters for preparing the basic protective solution are: temperature controlled at 20-25℃, stirring speed at 300-500 r / min, and time at 15-30 minutes; the specific parameters after adding the plasminogen activator are: temperature controlled at 2-8℃ and kept in the dark, stirring speed at 100-200 r / min, and time at 10-20 minutes.

8. The coating preparation method according to claim 6, characterized in that, The specific implementation method of step S200 is as follows: an ultrasonic atomizing micro-spraying device is used, and the atomized droplet particle size range during the spraying process is set to 20-50m; based on the preset specification of collecting 3mL of body fluid, the quantitative spraying loading volume of the single-tube composite functional mother liquor is 50-150L.

9. The coating preparation method according to claim 6, characterized in that, The specific process parameters for step S300 are as follows: Pre-freezing stage: The temperature of the plate is linearly reduced to -45 to -40℃ at a cooling rate of 1.0 to 2.5℃ / min, and held at a constant temperature for 120 to 180 minutes; First sublimation drying stage: The absolute pressure of the environment is reduced to 10-20 Pa, and the temperature of the plate is increased to -15 to -10℃ at a heating rate of 0.5-1.0℃ / min, and kept at a constant temperature for 300-480 minutes; Secondary analysis and drying stage: Maintain the absolute pressure of the environment at 10-20 Pa, raise the temperature of the plate to 20-25℃ at a rate of 1.0-1.5℃ / min, and keep it at a constant temperature for 180-240 minutes.

10. The coating preparation method according to claim 6, characterized in that, Following step S300, the following post-processing steps are also included: S400, in a nitrogen-protected production line with a purity of 99.9%, establishes a corresponding internal negative pressure environment according to the preset body fluid collection volume of the tube, and presses the tube cap and sealing component onto the top of the tube to complete the airtight sealing. S500 sterilizes the finished tubes with cobalt-60 radiation by penetrating irradiation after the airtight sealing is completed, and controls the absorbed dose range of 8 to 15 kGy during the irradiation process.

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