A method for preparing a multi-modal leukocyte filtration membrane

Abstract

The application discloses a preparation method of a multi-mode leukocyte filtering membrane. The application removes leukocytes with a large pore diameter by applying size exclusion mechanism to a coarse filtering layer, removes most of the leukocytes by utilizing charge interaction mechanism to a middle layer, and further removes the remaining leukocytes by adopting deep filtering mechanism to a final filtering layer. The synergistic effect of the three functional layers ensures that the leukocyte removal rate can meet the standard even under the condition of a large pore diameter, and the red blood cell recovery rate is improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of leukocyte filtration, and particularly relates to a preparation method of a multi-mode leukocyte filtration membrane. BACKGROUND

[0002] The application background of leukocyte filters is very wide, which can not only improve the safety of blood transfusion, but also is involved in the treatment of specific diseases and many other fields. In the process of blood transfusion, leukocyte filters can remove leukocytes in blood, reduce adverse reactions related to blood transfusion, such as febrile non-hemolytic transfusion reactions. In addition, it can also remove specific leukocyte subgroups to help regulate the immune response of patients.

[0003] In the actual filtration process, most leukocyte-removing filters take non-woven fabric as the core filter material and use the size exclusion principle to filter leukocytes. Since the leukocyte pore size is larger than the pore size of the non-woven filter membrane, the leukocytes are trapped outside the filter membrane and are successfully removed; and the red blood cells can pass through the smaller pores due to their deformability, so that the red blood cells can be recovered. At present, the leukocyte removal rate of leukocyte filters has reached more than 99.95%, and the red blood cell recovery rate is between 85-90%. However, in the face of the problem of gradually scarce blood resources, we need to further improve the red blood cell recovery rate. In order to solve this problem, researchers have taken some measures to improve the red blood cell recovery rate. For example, the patent KR1020130022815 “Biocompatible multi-layer filter and method of making the same” uses a leukocyte-removing filter composed of a combination of a plurality of biocompatible microporous fiber layers and nanofiber layers to avoid hemolysis and thus improve the red blood cell recovery rate. The patent CN201611031713.X “Leukocyte-removing filter for improving red blood cell recovery rate and use method thereof” uses 0.9% sodium chloride solution to soak the leukocyte-removing filter membrane for a period of time, uses 0.9% sodium chloride injection with substantially the same osmotic pressure as the blood to be filtered to avoid hemolysis and thus improve the red blood cell recovery rate. The patent KR1020080081768 “Filter and method of manufacturing the same for removing white particles” uses a chitosan-polyvinyl alcohol composite nanofiber web for filtration, avoids the risk of adding new substances for hydrophilic modification and blood compatibility modification, reduces the red blood cell destruction rate, and improves the red blood cell recovery rate. However, in actual blood filtration, the proportion of hemolysis and blood cell fragmentation is not large, and the effect of these measures on the improvement of the red blood cell recovery rate is limited. SUMMARY

[0004] The purpose of the present application is to solve the problems existing in the prior art and provide a preparation method of a multi-mode leukocyte filtration membrane.

[0005] In order to achieve the above object, the present application adopts the following technical scheme: A preparation method of a multi-mode leukocyte filter membrane, comprising the following steps,

[0006] S1: preparation of a coarse filter layer filter membrane,

[0007] 0.5-10% of the modifier is weighed in a three-necked flask, 90-99.5% of pure water is added, a constant-temperature water bath is used for stirring at 50-90°C for 60-300 minutes, a hydrophilic modifier solution is obtained after complete dissolution, then a coarse filter layer non-woven fabric with a pore size of 12-20μm is placed in the hydrophilic modifier solution for soaking for 5-30 minutes, an oven is used for solidification at 80-120°C for half an hour, then water is used for washing for 3 times, ultrasonic washing is performed for 15 minutes, and drying is performed to obtain a hydrophilic coarse filter layer filter membrane;

[0008] S2: preparation of an intermediate layer filter membrane,

[0009] 0.5-10% of the monomer and 90-99.5% of pure water are added to a three-necked flask, a constant-temperature water bath is used for stirring at 50-90°C for 60-300 minutes, a hydrophilic monomer solution is obtained after complete dissolution, then 2-20% of the initiator is added to the three-necked flask, a magnetic stirrer is used for stirring, and the initiator solution is obtained by heating at 50-80°C for 5-60 minutes during the stirring process; then a non-woven fabric with a pore size of 8-14μm is placed in the hydrophilic monomer solution for soaking for 5-30 minutes, and then placed in the initiator solution for soaking for 5-30 minutes, an oven is used for solidification at 70-100°C for half an hour, then water is used for washing for 3 times, ultrasonic washing is performed for 15 minutes, and drying is performed to obtain a hydrophilic positive intermediate layer filter membrane;

[0010] S3: preparation of a final filter layer filter membrane,

[0011] 2-20% of 2-methacryloyloxyethyl phosphorylcholine and 2-10% of potassium persulfate are weighed, mixed with 70-96% of water, and reacted at 45-80°C for 30-120 minutes, then 0.2-5% of a thickening agent is added, and stirring is continued for 60-360 minutes to obtain a hydrophilic modifier solution; then a non-woven fabric with a pore size of 8-11μm is placed in the hydrophilic modifier solution for soaking for 5-30 minutes, an oven is used for solidification at 80-120°C for half an hour, then water is used for washing for 3 times, ultrasonic washing is performed for 15 minutes, and drying is performed to obtain a hydrophilically modified final filter layer filter membrane.

[0012] As a further description of the above technical scheme:

[0013] The modifier in the step S1 is one of polyvinyl alcohol, chitosan, polyacrylic acid, polyvinylpyrrolidone and polyethylene glycol.

[0014] As a further description of the above technical scheme:

[0015] The monomer in the step S2 is one of 2-acetylamino acrylate and diethylaminoethyl methacrylate.

[0016] As a further description of the above technical solution:

[0017] The initiator in the step S2 is one of ammonium persulfate and potassium persulfate.

[0018] As a further description of the above technical solution:

[0019] The thickening agent in the step S3 is one of polyvinylpyrrolidone and polyvinyl alcohol.

[0020] As a further description of the above technical solution:

[0021] The non-woven fabric in the step S1, the non-woven fabric in the step S2 and the non-woven fabric in the step S3 are one of PBT, PET, PP, PA and PVC.

[0022] The present application has the following beneficial effects:

[0023] Compared with the prior art, the present application adopts a coarse filter layer, an intermediate layer and a final filter layer. The coarse filter layer removes leukocytes with a larger pore size by using size exclusion mechanism; the intermediate layer removes most of the leukocytes by using charge interaction mechanism; and the final filter layer further removes the remaining leukocytes by using deep filtration mechanism. The synergistic effect of the three functional layers ensures that the leukocyte removal rate can meet the standard even under large pore size conditions, and the red blood cell recovery rate is improved. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 The figure is a pore size distribution diagram of the coarse filter layer in the embodiment 1 of the present application;

[0025] Figure 2 The figure is a pore size distribution diagram of the intermediate layer in the embodiment 1 of the present application;

[0026] Figure 3 The figure is a pore size distribution diagram of the final filter layer in the embodiment 1 of the present application;

[0027] Figure 4 The figure is a pore size distribution diagram of the coarse filter layer in the embodiment 2 of the present application;

[0028] Figure 5 The figure is a pore size distribution diagram of the intermediate layer in the embodiment 2 of the present application;

[0029] Figure 6 The figure is a pore size distribution diagram of the final filter layer in the embodiment 2 of the present application;

[0030] Figure 7 The figure is a pore size distribution diagram of the coarse filter layer in the embodiment 3 of the present application;

[0031] Figure 8 The pore size distribution graph of the intermediate layer in Example 3 of the present application;

[0032] Figure 9 The pore size distribution graph of the final filter layer in Example 3 of the present application. DETAILED DESCRIPTION

[0033] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without creative labor fall within the protection scope of the present application.

[0034] Example 1:

[0035] S1: Preparation of the filter membrane of the coarse filter layer,

[0036] 1% polyvinyl alcohol was weighed in a three-necked flask, and 99% pure water was added. A constant-temperature water bath was used for stirring at 90°C for 150 minutes. After complete dissolution, a 1% hydrophilic modifier solution was obtained. Then, the coarse filter layer non-woven fabric PBT with a pore size of 14.87 μm was immersed in the hydrophilic modifier solution for 10 minutes, and an oven was used for curing at 80°C for half an hour. After washing with water for 3 times, ultrasonic washing for 15 minutes, and drying, the hydrophilic coarse filter layer filter membrane was obtained.

[0037] S2: Preparation of the filter membrane of the intermediate filter layer,

[0038] 2% diethylaminoethyl methacrylate and 96% pure water were added to a three-necked flask, and a constant-temperature water bath was used for stirring at 80°C for 120 minutes. After complete dissolution, a hydrophilic monomer solution was obtained. Then, 2% potassium persulfate was added to the three-necked flask, and a magnetic stirrer was used for stirring. During the stirring process, heating was performed at 60°C for 30 minutes to obtain an initiator solution. Then, the intermediate filter layer non-woven fabric PET with a pore size of 11.663 μm was immersed in the hydrophilic monomer for 15 minutes, and then immersed in the initiator solution for 15 minutes. An oven was used for curing at 80°C for half an hour. After washing with water for 3 times, ultrasonic washing for 15 minutes, and drying, the hydrophilic positive intermediate filter layer filter membrane was obtained.

[0039] S3: Preparation of the filter membrane of the final filter layer,

[0040] Take 2% of 2-methacryloyloxyethylphosphorylcholine and 2% of potassium persulfate, mix with 97% of water, react at 70°C for 60 minutes, then add 1% of polyvinylpyrrolidone, continue stirring for 60 minutes to obtain a hydrophilic modification solution, then put the 9.291 μm pore size final filter layer non-woven fabric PBT into the hydrophilic modifier solution for 5 minutes, use an oven to cure at 90°C for half an hour, wash with water 3 times, ultrasonic washing for 15 minutes, and dry to obtain the hydrophilic modified final filter layer filter membrane.

[0041] Example 2: The same as example 1, except that the types and pore sizes of each layer of the filter membrane are different. The coarse filter layer substrate is PET, with a pore size of 14.110 μm. The middle filter layer substrate is PBT, with a pore size of 10.354 μm. The final filter layer filter substrate is PBT, with a pore size of 9.291 μm.

[0042] Example 3: The same as example 1, except that the types and pore sizes of each layer of the filter membrane are different. The coarse filter layer substrate is PBT, with a pore size of 14.81 μm. The middle filter layer substrate is PBT, with a pore size of 10.354 μm. The final filter layer filter substrate is PBT, with a pore size of 9.291 μm.

[0043] Test Example

[0044] 1. As Figures 1-9 is the pore size distribution diagram of the non-woven filter membrane.

[0045] 2. Assembly test of leukocyte filter

[0046] Use a slicer to cut the modified filter membrane into a leukocyte filter membrane with a diameter of 6 cm. The total number of filter membranes is 8 layers, from top to bottom, coarse filter layer filter membrane 2 layers, middle layer filter membrane 3 layers, and final filter layer filter membrane 3 layers. The thickness of each layer of the coarse filter layer and the middle layer is between 0.300-0.350 μm. The thickness of each layer of the final filter layer filter membrane is 0.650-0.700 μm. Use collected human blood to filter out white blood cells, and each time 400 ml of blood is filtered.

[0047] 3. Blood filtration results

[0048] The content of blood cells in blood is usually determined by a blood routine instrument, which can quickly measure the number of red blood cells (RBC) and white blood cells (WBC) in blood. However, after the blood is treated by a leukocyte filter, the content of white blood cells may be reduced to a very low level. In this case, the traditional blood routine instrument may not be able to provide accurate enough measurement results. Therefore, we turn to the microscopic counting method in YY0329-2009 Disposable Leukocyte Removal Filter to calculate the remaining white blood cell content.

[0049] Leukocyte removal filter remaining white blood cell number calculation formula:

[0050] In the formula:

[0051] LR - the number of residual white blood cells, unit: pieces;

[0052] n - the average value of the white blood cell count of 3 tubes of samples, unit: pieces;

[0053] V - the volume of blood components after filtration, unit: milliliters;

[0054] Vs - the volume of blood sample for counting, unit: microliters;

[0055] 103 - conversion factor, 1 mL = 103 μL

[0056] Red blood cell recovery rate calculation formula;

[0057] In the formula:

[0058] ER - red blood cell recovery rate, %;

[0059] Rq - red blood cell concentration of blood sample before filtration, hemocytometer reading value;

[0060] Rh - red blood cell concentration of blood sample after filtration, hemocytometer reading value;

[0061] Vq - the volume of blood sample before filtration, unit: milliliters;

[0062] Vh - the volume of blood sample after filtration, unit: milliliters.

[0063] Table 1: Summary of blood filtration test data

[0064] Name Example 1 Example 2 Example 3 Domestic filter Volume of blood before filtration (mL) 400 400 400 400 Volume of blood after filtration (mL) 378 381 380 371 Number of primitive blood leukocytes (10 9 / L) 5.52 5.62 5.47 5.98 6 ​​ 0.87 0.73 0.82 0.91 Leukocyte removal rate (%) 99.984 99.987 99.985 99.985 Number of proerythroblasts (10 12 / L) 3.72 3.62 3.79 3.59 <![CDATA[滤后红细胞个数(10 12 / L)]]> 3.73 3.60 3.79 3.42 Red blood cell recovery rate (%) 94.75 95.78 95.00 88.36

[0065] By comparing and analyzing the filtration data of Examples 1, 2, 3 and the competitor, we found that these examples meet the requirements of the standard of YY0329-2009 Disposable Leukocyte Removal Filter in terms of white blood cell removal rate and red blood cell recovery rate. Specifically, the white blood cell removal rate of the examples is comparable to that of domestic filters. However, in terms of blood recovery volume and red blood cell recovery rate, the performance of the examples is more outstanding. The specific improvements are that the blood volume after filtration increases by 10 milliliters, and the red blood cell recovery rate increases from 88.36% to 95.78%.

[0066] Finally, it should be noted that the above only describes the preferred embodiments of the present application and is not intended to limit the present application. Although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art will appreciate that the technical solutions described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalent features, and any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for preparing a multi-mode leukocyte filtration membrane, characterized in that: The following steps are included: S1: Preparation of coarse filter membrane, Weigh 0.5-10% of the modifier into a three-necked flask, add 90-99.5% pure water, and stir in a constant temperature water bath at 50-90°C for 60-300 minutes to obtain a hydrophilic modifier solution after complete dissolution. Then, place a coarse filter layer non-woven fabric with a pore size of 12-20 μm in the hydrophilic modifier solution and soak it for 5-30 minutes. Use an oven to cure it at 80-120°C for half an hour, then wash it with water three times, ultrasonically wash it for 15 minutes, and dry it to obtain a hydrophilic coarse filter layer membrane. S2: Preparation of the intermediate filter membrane, Add 0.5-10% monomer and 90-99.5% pure water to a three-necked flask, stir at 50-90°C for 60-300 minutes in a constant temperature water bath, and obtain a hydrophilic monomer solution after complete dissolution. Then, add 2-20% initiator to the three-necked flask, stir with a magnetic stirrer, and heat at 50-80°C for 5-60 minutes during stirring to obtain an initiator solution. Then, place an intermediate layer non-woven fabric with a pore size of 8-14 μm in the hydrophilic monomer solution and soak for 5-30 minutes, then in the initiator solution and soak for 5-30 minutes, use an oven to cure at 70-100°C for half an hour, wash with water three times, ultrasonically wash for 15 minutes, and dry to obtain a hydrophilic positively charged intermediate layer filter membrane. S3: Preparation of final filter membrane, 2-20% of 2-methacryloyloxyethyl phosphorylcholine and 2-10% of potassium persulfate are weighed, mixed with 70-96% of water, and reacted at 45-80°C for 30-120 minutes. Then, 0.2-5% of a thickener is added, and stirring is continued for 60-360 minutes to obtain a hydrophilic modifier solution. Then, a final filter layer non-woven fabric with a pore size of 8-11 μm is placed in the hydrophilic modifier solution and soaked for 5-30 minutes. The fabric is cured in an oven at 80-120°C for half an hour, washed with water three times, ultrasonically washed for 15 minutes, and dried to obtain a hydrophilically modified final filter layer membrane.

2. The method for preparing a multi-mode leukocyte filtration membrane according to claim 1, characterized in that: The modifier in step S1 is one of polyvinyl alcohol, chitosan, polyacrylic acid, polyvinyl pyrrolidone and polyethylene glycol.

3. The method for preparing a multi-mode leukocyte filtration membrane according to claim 1, characterized in that: The monomer in step S2 is one of 2-acetamidoacrylic acid and diethylaminoethyl methacrylate.

4. The method for preparing a multi-mode leukocyte filtration membrane according to claim 1, wherein: The initiator in step S2 is one of ammonium persulfate and potassium persulfate.

5. The method for preparing a multi-mode leukocyte filtration membrane according to claim 1, characterized in that: The thickener in step S3 is one of polyvinyl pyrrolidone and polyvinyl alcohol.

6. The method for preparing a multi-mode leukocyte filtration membrane according to claim 1, characterized in that: The coarse filter layer non-woven fabric in step S1, the intermediate filter layer non-woven fabric in step S2 and the final filter layer non-woven fabric in step S3 are all selected from the group consisting of PBT, PET, PP, PA and PVC.

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