In-vitro dynamic hemolysis evaluation method for blood adsorption material with high specific surface area

Abstract

The invention belongs to the technical field of detection, and discloses an evaluation method for in-vitro dynamic hemolysis of a blood adsorption material with a high specific surface area. A chromatographic column is used for simulating a blood adsorption product, the column height of the chromatographic column and the blood adsorption product, the blood retention time and the total contact time of unit blood and an adsorbent are controlled to be the same, and then the hemolysis rate of an adsorption material to be tested in the chromatographic column is tested. According to the evaluation method of some examples, the hemolysis rate data of the adsorption products of different manufacturers are tested by adopting an equal-proportion reduction dynamic simulation contact method and are very close to the hemolysis rate data measured by adopting a whole-column dynamic contact method, and the hemolysis effect of the real blood adsorption products can be reflected.

Description

Technical Field

[0001] The invention belongs to the field of detection technology, and in particular relates to an evaluation method for in vitro dynamic hemolysis of a high specific surface blood adsorption material. Background Art

[0002] The national standard GB / T16886.4-2022 "Biological Evaluation of Medical Devices Part 1: Evaluation and Testing in the Risk Management Process" issued by the National Medical Products Administration and the National Standardization Administration in 2022 clearly states that biocompatibility is defined as: the ability of a medical device or material to adapt to host responses in specific applications. Blood compatibility is an important part of biocompatibility evaluation. The interaction between medical devices and blood or blood components may cause multiple effects on human blood, organs, tissues or device performance, which is directly related to the safety and effectiveness of the clinical application of such devices. We hope to develop extracorporeal circulation medical device products with better biocompatibility and blood compatibility to reduce adverse reactions in the human body during their clinical use. Therefore, preclinical evaluation and testing of device-blood interactions are very important contents in the biological evaluation of medical devices.

[0003] Blood compatibility includes cellular level reactions such as thrombosis, leukocyte activation, and hemolysis after the material comes into contact with blood; protein level reactions such as activation of the coagulation system, activation of the fibrinolytic system, and complement activation; and a series of molecular level reactions such as ADP and platelet factor release. It covers a wide range and has a complex reaction mechanism, and needs to be evaluated from different levels and levels. Among them, the hemolysis test is a common and important screening test for the blood compatibility test of medical devices. At present, the international standards related to the hemolysis test of medical devices include ISO 10993-4, ASTM F 756, and the domestic standards include GB / T 16886.4, GB / T16175, GB / T 14233.2 and YY / T 1651.1. The hemolytic effect of medical devices is generally divided into osmotic pressure-mediated hemolysis, material-mediated hemolysis, and mechanical force-mediated hemolysis. Material-mediated hemolysis tests can be divided into direct contact and indirect contact methods. GB / T 16175 and GB / T 14233.2 provide the test methods for direct contact methods, while YY / T 1651.1 provides the test methods for direct contact and indirect contact methods. However, the above in vitro contact methods, whether direct or indirect, do not take into account the hemolytic potential of the mechanical force of the device under clinical hemodynamic conditions, and for devices with high specific surface areas (specific surface area not less than 800 m 2 / g) of blood adsorption materials, there is a certain deviation between the experimental contact ratio and the actual clinical contact ratio. At the same time, it is not considered that high specific surface area materials have a certain adsorption effect on blood components such as hemoglobin, and it is often difficult to obtain the actual hemolysis situation after the interaction between the device and blood.

[0004] In order to more truly reflect the hemolytic reaction of the clinical use of the device, the ideal in vitro model is that the finished blood adsorption product is connected to the blood pump through the extracorporeal circulation pipeline as a circulation pathway, and the whole blood is perfused at the clinical flow rate and treatment time. However, the disadvantages of using this model for evaluation are: the test samples need to be made into finished products, the materials in the laboratory research and development stage cannot be quickly tested, the blood treatment dose is large, and the investment cost is high. Therefore, it is urgent to establish an in vitro hemolysis evaluation method that can more equivalently simulate the contact ratio of high specific surface area blood adsorption materials with blood during clinical use, more accurately indicate the hemolytic performance of the material, and further save the evaluation cost under the premise of rapid and easy testing. Summary of the invention

[0005] The object of the present invention is to overcome at least one disadvantage of the prior art and to provide a method for evaluating in vitro dynamic hemolysis of a high specific surface area blood adsorption material.

[0006] The technical solution adopted by the present invention is: An in vitro dynamic hemolysis evaluation method for a high specific surface area blood adsorption material comprises the following steps: A chromatographic column is used to simulate a blood adsorption product. The chromatographic column is filled with a fill volume V' of the adsorbent to be tested as a test sample. 0 'The test blood is added to the conical flask, and then connected to the circulation pipeline, chromatography column, and roller pump. The roller pump is turned on and the volume flow rate v mL 'Cycle response time T', where: , , , , where D' is the diameter of the chromatography column, H is the column height of the adsorbed product, v cm is the linear flow rate of adsorption product, T 0 is the blood retention time, t is the total time that the adsorbed product unit blood contacts the adsorbent, V 0 ' is the amount of blood used in the chromatography column, D' is a preset value, H, T 0 , t are known parameters of the adsorbed product; Purified water was selected as the positive control and saline as the negative control, added to the conical flask, and then connected to the circulation pipeline, chromatography column, and roller pump. The chromatography column of the control group was not filled with adsorbent, and the roller pump was turned on at the calculated volume flow rate v. mL 'Cycle corresponding time T'; At the end of the cycle, all the liquids in the circulation system were collected in conical flasks, and an appropriate amount of liquid was centrifuged to obtain the supernatant. The absorbance of the supernatant was measured at a wavelength of 545 nm using an ultraviolet spectrophotometer to calculate the hemolysis rate HR. , where: HR--hemolysis rate of blood adsorption material, % A--absorbance of test sample group; B--absorbance of negative control group; C--absorbance of positive control group.

[0007] In some examples of the evaluation method, the test blood is diluted anticoagulated rabbit blood, the test sample group and the negative control group are diluted with physiological saline, and the positive control group is diluted with purified water.

[0008] In some examples of the evaluation method, the mass ratio of the adsorbent to be tested to the volume of physiological saline used for dilution of the test sample group is 0.2-0.5 g / mL, and the volume ratio of the diluted anticoagulated rabbit blood to the physiological saline is (1-75):50. Preferably, the mass ratio of the adsorbent to be tested to the volume of physiological saline is 0.5 g / mL.

[0009] In some examples of the evaluation method, the amount of test blood V 0 ' is 5 to 12.5 times the mass of the adsorbent to be tested. Preferably, the amount of test blood V 0 ' is 5 times the mass of the adsorbent to be tested.

[0010] In some examples of the evaluation method, the temperature of the blood sample is controlled at 37±1°C during the circulation process.

[0011] In some examples of the evaluation method, the centrifugation operation is specifically centrifugation at 800 g for 5 min.

[0012] In some examples of the evaluation method, after the circulation line is connected, the air in the system is exhausted with physiological saline to perform system pre-filling.

[0013] In some examples of evaluation methods, before the cycle test begins, the roller pump is turned on to discharge the pre-filled saline in the system, and the discharged liquid volume is recorded to be consistent with the total volume of the chromatography column and the pipeline. The test is stopped when the color of the liquid at the outlet is close to the color of the liquid in the conical flask.

[0014] In some examples of the evaluation method, the specific surface area of ​​the adsorbent to be tested is not less than 800 m 2 / g.

[0015] In some examples of the evaluation method, the conical bottle is rinsed with saline before adding the blood sample.

[0016] The above technical features can be combined arbitrarily without conflict.

[0017] The beneficial effects of the present invention are: The evaluation methods of some examples of the present invention use the proportional reduction dynamic simulation contact method to test the hemolysis rate data of adsorption products from different manufacturers. The data are very close to the hemolysis rate data measured by the whole column dynamic contact method, which can reflect the hemolysis effect of the real blood adsorption product. However, the hemolysis rate test results of the direct contact method are quite different from those of the proportional reduction dynamic simulation contact method and the whole column dynamic contact method, indicating that the direct contact method has a large deviation in reflecting the real hemolysis rate of the blood adsorption product in clinical application.

[0018] The evaluation methods of some examples of the present invention are different from the direct contact method of static incubation in a water bath, which completely ignores the mechanically mediated hemolysis during clinical use of the product or the additional hemolysis caused by the mechanical force applied by constant temperature oscillation incubation. The proportionally reduced dynamic simulated contact method is used. The testing process is closer to the actual clinical state and can reflect the hemolysis mediated by mechanical force and biochemical factors when the adsorption product is used.

[0019] The evaluation method of some examples of the present invention adopts a proportionally reduced dynamic simulated contact method. The filling amount of the adsorbent and the amount of blood used in the test are significantly reduced compared with the direct use of the blood adsorption product, and the blood flow rate is also significantly reduced, while the difference in the test data is not obvious, which can well simulate the clinical use situation. The cost and difficulty of the test are greatly reduced, and it is an effective simulation test method. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is a schematic diagram of the system structure used in the evaluation method of the present invention. DETAILED DESCRIPTION

[0021] For blood adsorption products, the amount of adsorption material V, the product column height H (or column cross-sectional diameter D), and the linear flow rate v cm (or clinical flow rate v mL ) and the total product treatment time T are determined, and the human blood volume V under clinical use conditions 0 It is determined based on the normal blood volume of a normal adult, which is 4.5L. If the population using the blood adsorption product has special requirements, V 0 The total time t that a unit of blood of the adsorption product contacts the adsorbent can also be calculated based on the known parameters of the adsorption product, i.e. The product column height can also be determined based on known parameters of the adsorbed product. , linear flow rate .

[0022] The technical solution of the present invention is further explained below in conjunction with experiments.

[0023] Example 1: Testing the hemolysis rate of different adsorption products by the dynamic simulated contact method with proportional reduction (1) Chromatography column preparation Select blood adsorption products from different manufacturers and determine the product column height H, diameter D, and clinical flow rate v mL , total product treatment time T; Select the proportional reduction model corresponding to the adsorption product, with a diameter D' of 15 mm. According to the parameters of the blood adsorption product, determine the column height H, the adsorbent filling volume V', and the column volume flow rate v mL ' ， Blood volume for chromatography column V 0 ', and cycle time T'; the comparison of key parameters of different adsorption products and their proportional reduction models is shown in Table 1.

[0024] Fill the chromatography column according to the amount of adsorbent, fix it on the bracket, connect both ends to the shortest clean circulation pipeline, pre-flush it from bottom to top with saline, and fill the pipeline and chromatography column with saline for use.

[0025] Table 1. Comparison of key parameters of proportional reduction models of different adsorption products

[0026] (2) Preparation of blood for experiment: Fresh 3.2% sodium citrate anticoagulated rabbit blood was diluted with normal saline at a ratio of 4:5 and mixed to prepare diluted anticoagulated rabbit blood; (3) Sample preparation: Test sample: Take a disposable blood perfusion device and pre-flushed it from bottom to top with normal saline according to the instructions. After the pre-flushing, remove excess water by filtration to keep the adsorbent moist. It has been verified that when the adsorbent is filled in a 15 mm inner diameter chromatography column to reach the same column height as the product, the adsorbent mass of commercial product 1, commercial product 2, and commercial product 3 is approximately 9, 10, and 8 g. According to the ratio of adsorbent mass to extract volume of 0.5 g / mL specified in GB / T 14233.2-2005 standard, take 0.9% sodium chloride injection in proportion for use; Negative control: Take 20 mL of 0.9% sodium chloride injection as the negative control group; Positive control: Take 20 mL of purified water as the positive control group.

[0027] (4) Cycle test: Add the corresponding liquid (normal saline for the test sample group and negative control group, purified water for the positive control group) with a volume to adsorbent mass ratio of 0.5 g / mL to the conical flask rinsed with normal saline, gently mix the diluted anticoagulated rabbit blood, and add it to the conical flask (the volume ratio of the diluted anticoagulated rabbit blood to the corresponding liquid is 3:2). Connect the conical flask to the circulation pipeline, and turn on the roller pump to discharge the normal saline pre-filled in the circulation system. According to preliminary verification, the total volume of the empty chromatography column and pipeline is about 40mL. Therefore, a measuring cylinder was used to record the volume of discharged liquid in this experiment. The negative and positive control groups stopped when they reached 40.0mL, and the test group stopped when it reached about 30.0mL (the color of the liquid flowing out of the outlet was close to the color of the blood in the conical flask). The conical flask containing the experimental blood was placed in a constant temperature water bath at 37℃±1℃, and the circulation was turned on. The model flow rate and circulation time were proportionally reduced for each product for dynamic contact (KCM150: 16.84 mL / min, 17.82 min; commercial product 1: 19.29 mL / min, 17.97 min; commercial product 2: 16.72 mL / min, 15.55 min). (5) Hemolysis rate determination: After the cycle was completed, all the liquid in the system was collected in a conical flask and mixed evenly. A 5 mL blood sample was taken and centrifuged at 800 g for 5 min in a 15 mL centrifuge tube, and the supernatant was taken. The absorbance of the supernatant was measured at a wavelength of 545 nm using an ultraviolet-visible spectrophotometer to calculate the hemolysis rate.

[0028] Comparative Example 1: Testing the hemolysis rate of different adsorption products by direct contact method (1) Sample preparation: Test sample: Use 50 mL centrifuge tubes to take three kinds of adsorption resins (9, 10, 8 g) of the same batch and mass of the dynamic model contact group in equal proportion, and add 18, 20, and 16 mL of normal saline respectively according to the ratio of adsorbent mass to extract volume of 0.5 g / mL; Negative control: Use a 50 mL centrifuge tube and add 20.0 mL of normal saline; Positive control: Use a 50 mL centrifuge tube and add 20.0 mL of purified water.

[0029] (2) Cycle test: ① Static incubation in a water bath: Place all test tubes in a 37℃±1℃ constant temperature water bath and incubate for 30 min; after 30 min, use a pipette to add the corresponding volume of diluted anticoagulant rabbit blood to each centrifuge tube at a ratio of adsorbent mass / extract volume / anticoagulant diluted rabbit blood volume of 0.5 g / 10 mL / 0.2 mL, gently invert the test tubes upside down to mix, and place all test tubes in a 37℃±1℃ constant temperature water bath again and incubate for 60 min.

[0030] ② Constant temperature shaking incubation: Place all test tubes in a constant temperature shaking incubator at 37℃±1℃ for 30 min at a rotation speed of 60 rpm; after 30 min, use a pipette to add the corresponding volume of diluted anticoagulant rabbit blood to each centrifuge tube at the ratio of adsorbent mass / extract volume / anticoagulant diluted rabbit blood volume of 0.5 g / 10 mL / 0.2 mL, gently invert the test tubes upside down to mix, and place all test tubes in a constant temperature shaking incubator at 37℃±1℃ for incubation for 60 min at a rotation speed of 60 rpm.

[0031] (3) Hemolysis rate determination: After the incubation, all solutions were transferred to appropriate centrifuge tubes and centrifuged at 800 g for 5 min. The supernatant was taken and the absorbance was measured at a wavelength of 545 nm using a UV-visible spectrophotometer to calculate the hemolysis rate.

[0032] Comparative Example 2: Testing the hemolysis rate of different adsorption products by the whole column dynamic contact method Similar to Example 1, the complete disposable hemoperfusion devices, commercially available products 1, 2 and 3, were used for testing.

[0033] The hemolysis rate test results are shown in Table 2.

[0034] Table 2. Hemolysis rate test results of different evaluation methods

[0035] Through the test data of hemolysis rate, it can be found that: (1) The hemolysis rate data of adsorption products from different manufacturers measured by different test methods show the same change pattern, indicating that different test methods can reflect the relative trend of the hemolysis performance of adsorption products.

[0036] (2) The hemolysis rate data of adsorption products from different manufacturers were tested by using the proportionally reduced dynamic simulated contact method. The data were very close to the hemolysis rate data measured by the whole column dynamic contact method, which can reflect the hemolysis effect of real blood adsorption products.

[0037] (3) The difference in hemolysis rate test results between the direct contact method and the proportional dynamic simulated contact method and the whole column dynamic contact method is large, indicating that the direct contact method has a large deviation in reflecting the actual hemolysis rate of blood adsorption products in clinical applications.

[0038] (4) Unlike the direct contact method of static incubation in a water bath, which completely ignores the mechanically mediated hemolysis during clinical use of the product or the additional hemolysis caused by the mechanical force applied during constant temperature oscillation incubation, the proportionally reduced dynamic simulated contact method test process is closer to the actual clinical state and can reflect the hemolysis mediated by mechanical force and biochemical factors when the adsorption product is used.

[0039] (5) By using the proportionally reduced dynamic simulated contact method, the amount of adsorbent and blood used in the test are significantly reduced compared to the direct use of blood adsorption products, and the blood flow rate is also significantly reduced, while the difference in test data is not obvious, which can well simulate clinical use. The cost and difficulty of the test are greatly reduced, which is an effective simulation test method.

[0040] The above is a further detailed description of the present invention, which should not be regarded as a limitation on the specific implementation of the present invention. For ordinary technicians in the technical field to which the present invention belongs, simple deduction or replacement without departing from the concept of the present invention is within the protection scope of the present invention.

Claims

1. A method for evaluating in vitro dynamic hemolysis of a high specific surface area blood adsorption material, comprising the following steps: A chromatographic column is used to simulate a blood adsorption product. The chromatographic column is filled with a filling volume V' of the adsorbent to be tested as a test sample. The test blood with a volume of V0' is added to a conical flask, which is then circulated to the circulation pipeline, the chromatographic column, and the roller pump. The roller pump is turned on and the volume flow rate v is set. mL 'Cycle response time T', where: , , , , where D' is the diameter of the chromatography column, H is the column height of the adsorbed product, v cm is the linear flow rate of the adsorption product, T0 is the blood retention time, t is the total time that the unit blood of the adsorption product contacts the adsorbent, V0' is the blood volume used in the chromatography column, D' is a preset value, H, T0, and t are known parameters of the adsorption product; Purified water was selected as the positive control and saline as the negative control, added to the conical flask, and then connected to the circulation pipeline, chromatography column, and roller pump. The chromatography column of the control group was not filled with adsorbent, and the roller pump was turned on at the calculated volume flow rate v. mL 'Cycle corresponding time T'; At the end of the cycle, collect all the liquids in the circulation system in conical flasks, take an appropriate amount of liquid and centrifuge to obtain the supernatant, use an ultraviolet spectrophotometer to measure the absorbance of the supernatant at a wavelength of 545 nm, and calculate the hemolysis rate HR. , where: HR--hemolysis rate of blood adsorption material, % A--absorbance of test sample group; B--absorbance of negative control group; C--absorbance of positive control group.

2. The evaluation method according to claim 1, characterized in that: The test blood was diluted anticoagulated rabbit blood. The test sample group and the negative control group were diluted with physiological saline, and the positive control group was diluted with purified water.

3. The evaluation method according to claim 2, characterized in that: The mass ratio of the adsorbent to be tested to the volume of physiological saline used for dilution of the test sample group was 0.2-0.5 g / mL, and the volume ratio of the diluted anticoagulated rabbit blood to the physiological saline was (1-75):

50.

4. The evaluation method according to any one of claims 1 to 3, characterized in that: The amount V0' of the test blood is 5 to 12.5 times the mass of the adsorbent to be tested.

5. The evaluation method according to any one of claims 1 to 3, characterized in that: The temperature of the blood samples was controlled at 37±1°C during the circulation process.

6. The evaluation method according to any one of claims 1 to 3, characterized in that: The centrifugation operation was performed at 800 g for 5 min.

7. The evaluation method according to any one of claims 1 to 3, characterized in that: After connecting the circulation line, use physiological saline to exhaust the air in the system and pre-fill the system.

8. The evaluation method according to any one of claims 1 to 3, characterized in that: Before the cycle test begins, the roller pump is turned on to discharge the pre-filled physiological saline in the system, and the discharged liquid volume is recorded to be consistent with the total volume of the chromatography column and the pipeline. The test is stopped when the color of the liquid at the outlet is close to the color of the liquid in the conical flask.

9. The evaluation method according to any one of claims 1 to 3, characterized in that: The specific surface area of ​​the adsorbent to be tested is not less than 800 m 2 / g.

10. The evaluation method according to any one of claims 1 to 3, characterized in that: The conical flask was rinsed with physiological saline before adding the blood sample.

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