Preparation method of multi-mode leukocyte filtering membrane

Abstract

The invention discloses a preparation method of a multi-mode leukocyte filtering membrane. The multi-mode leukocyte filtering membrane adopts a rough filtering layer, a middle layer and a final filtering layer. The coarse filtering layer adopts a size exclusion mechanism to remove white blood cells with relatively large pore diameters; the middle layer utilizes a charge interaction mechanism to remove most white blood cells; and the final filtering layer adopts a deep filtering mechanism to further remove residual leukocytes. The synergistic effect of the three functional layers ensures that the white blood cell removal rate can meet the standard even under the condition of large aperture, and meanwhile, the recovery rate of red blood cells is improved.

Description

Technical Field

[0001] The invention relates to the technical field of leukocyte filtration, and in particular to a method for preparing a multi-mode leukocyte filtration membrane. Background Art

[0002] The application background of leukocyte filters is very wide. It can not only improve the safety of blood transfusion, but also involve many fields such as the treatment of specific diseases. During the blood transfusion process, leukocyte filters can remove white blood cells from the blood and reduce adverse reactions related to blood transfusion, such as febrile non-hemolytic transfusion reactions. In addition, it can also remove specific leukocyte subsets to help regulate the patient's immune response.

[0003] In the actual filtration process, most leukocyte removal filters use non-woven fabrics as the core filter material and use the size exclusion principle to filter leukocytes. Since the pore size of leukocytes is larger than that of the non-woven filter membrane, they are retained on the outside of the filter membrane and are successfully removed; while red blood cells can pass through smaller pores due to their own deformability, allowing red blood cells to be recovered. At present, the leukocyte removal rate of leukocyte filters has reached more than 99.95%, and the recovery rate of red blood cells is between 85-90%. However, in the face of the problem of the gradual scarcity of blood resources, we need to further improve the recovery rate of red blood cells. In order to solve this problem, researchers have taken some measures to improve the recovery rate of red blood cells. For example, patent KR1020130022815 "Biocompatible multilayer filter and its preparation method" uses a leukocyte removal filter composed of a combination of multiple microporous fiber layers and nanofiber layers with good biocompatibility to avoid hemolysis, thereby improving the recovery rate of red blood cells. Patent CN201611031713.X "A leukocyte-removing filter for improving red blood cell recovery rate and its use method" uses 0.9% sodium chloride solution to soak the leukocyte-removing filter membrane for a period of time, and uses 0.9% sodium chloride injection to have basically the same osmotic pressure as the blood to be filtered, thereby avoiding hemolysis problems and improving the red blood cell recovery rate. Patent KR1020080081768 "Filter and its manufacturing method for removing white particles" uses chitosan-polyvinyl alcohol composite nanofiber mesh for filtration, avoiding the risk of adding new substances to hydrophilic modification and blood compatibility modification, reducing the red blood cell destruction rate, and improving the red blood cell recovery rate. However, in actual blood filtration, the proportion of hemolysis and blood cell fragmentation is not large, and these measures have limited effect on improving the red blood cell recovery rate. Summary of the invention

[0004] The purpose of the present invention is to solve the shortcomings of the prior art and to propose a method for preparing a multi-mode leukocyte filtration membrane.

[0005] In order to achieve the above object, the present invention adopts the following technical scheme: a method for preparing a multi-mode leukocyte filtration membrane, comprising the following steps:

[0006] S1: Preparation of coarse filter membrane,

[0007] Weigh 0.5-10% of the modifier into a three-necked flask, add 90-99.5% pure water, use a constant temperature water bath to stir at 50-90°C for 60-300 minutes, and obtain a hydrophilic modifier solution after complete dissolution. Then, put a coarse filter layer non-woven fabric with a pore size of 12-20 μm into 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 3 times, ultrasonically wash it for 15 minutes, and dry it to obtain a hydrophilic coarse filter layer membrane;

[0008] S2: Preparation of the intermediate filter membrane,

[0009] Add 0.5-10% of monomer and 90-99.5% of pure water to a three-necked flask, stir at 50-90°C for 60-300 minutes using a constant temperature water bath, and obtain a hydrophilic monomer solution after complete dissolution; then add 2-20% of initiator to the three-necked flask, stir using a magnetic stirrer, and heat at 50-80°C for 5-60 minutes during the stirring process to obtain an initiator solution; then place the middle layer non-woven fabric with a pore size of 8-14 μm in the hydrophilic monomer solution for 5-30 minutes, then place it in the initiator solution for 5-30 minutes, use an oven to cure at 70-100°C for half an hour, then wash with water 3 times, ultrasonically wash for 15 minutes, and dry to obtain a hydrophilic positively charged middle layer filter membrane;

[0010] S3: Preparation of final filter membrane,

[0011] 2-20% of 2-methacryloyloxyethyl phosphorylcholine and 2-10% of potassium persulfate are weighed, mixed with 70-96% of water, 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, cured in an oven at 80-120°C for half an hour, washed with water for 3 times, ultrasonically washed for 15 minutes, and dried to obtain a hydrophilically modified final filter layer filter membrane.

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

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

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

[0015] The monomer in step S2 is one of 2-acetamidoacrylic acid and diethylaminoethyl methacrylate.

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

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

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

[0019] The thickener in step S3 is one of polyvinyl pyrrolidone and polyvinyl alcohol.

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

[0021] The coarse filter layer nonwoven fabric in step S1, the intermediate filter layer nonwoven fabric in step S2 and the final filter layer nonwoven fabric in step S3 are all selected from the group consisting of PBT, PET, PP, PA and PVC.

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

[0023] Compared with the prior art, the present invention adopts a coarse filter layer, an intermediate layer and a final filter layer. The coarse filter layer uses a size exclusion mechanism to remove white blood cells with larger pores; the intermediate layer uses a charge interaction mechanism to remove most white blood cells; and the final filter layer uses a deep filtration mechanism to further remove the remaining white blood cells. The synergistic effect of these three functional layers ensures that the white blood cell removal rate can meet the standard even under large pore conditions, while improving the recovery rate of red blood cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the pore size distribution diagram of the coarse filter layer in Example 1 of the present invention;

[0025] Figure 2 This is the pore size distribution diagram of the middle layer of Example 1 of the present invention;

[0026] Figure 3 This is the pore size distribution diagram of the final filter layer in Example 1 of the present invention;

[0027] Figure 4 This is the pore size distribution diagram of the coarse filter layer in Example 2 of the present invention;

[0028] Figure 5 This is the pore size distribution diagram of the middle layer of Example 2 of the present invention;

[0029] Figure 6 This is the pore size distribution diagram of the final filter layer in Example 2 of the present invention;

[0030] Figure 7 This is the pore size distribution diagram of the coarse filter layer in Example 3 of the present invention;

[0031] Figure 8 This is the pore size distribution diagram of the middle layer of Example 3 of the present invention;

[0032] Fig. 9 This is the pore size distribution diagram of the final filter layer in Example 3 of the present invention. DETAILED DESCRIPTION

[0033] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0034] Embodiment 1:

[0035] S1: Preparation of coarse filter membrane,

[0036] Weigh 1% polyvinyl alcohol in a three-necked flask, and add 99% pure water. Stir at 90°C in a constant temperature water bath for 150 minutes. After complete dissolution, a 1% hydrophilic modifier solution is obtained. Then, a coarse filter layer non-woven fabric PBT with a pore size of 14.87μm is placed in the hydrophilic modifier solution and soaked for 10 minutes. Use an oven to cure at 80°C for half an hour, wash with water 3 times, ultrasonically wash for 15 minutes, and dry to obtain a hydrophilic coarse filter layer membrane.

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

[0038] Add 2% diethylaminoethyl methacrylate and 96% pure water to a three-necked flask, stir at 80°C for 120 minutes in a constant temperature water bath, and obtain a hydrophilic monomer solution after complete dissolution. Then add 2% potassium persulfate to the three-necked flask, stir with a magnetic stirrer, heat and react at 60°C for 30 minutes during stirring to obtain an initiator solution, and then soak the middle filter layer non-woven fabric PET with a pore size of 11.663μm in the hydrophilic monomer for 15 minutes, and then soak in the initiator solution for 15 minutes, and use an oven to cure at 80°C for half an hour, wash 3 times with water, ultrasonically wash for 15 minutes, and dry to obtain a hydrophilic positively charged middle filter layer membrane.

[0039] S3: Preparation of final filter membrane,

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

[0041] Example 2: Same as Example 1, except that the types and pore sizes of the filter membranes in each layer are different. The base material of the coarse filter layer is PET, with a pore size of 14.110 μm. The base material of the medium filter layer is PBT, with a pore size of 10.354 μm. The base material of the final filter layer is PBT, with a pore size of 9.291 μm.

[0042] Example 3: Same as Example 1, except that the types and pore sizes of the filter membranes in each layer are different. The base material of the coarse filter layer is PBT, with a pore size of 14.81 μm. The base material of the medium filter layer is PBT, with a pore size of 10.354 μm. The base material of the final filter layer is PBT, with a pore size of 9.291 μm.

[0043] Test Case

[0044] 1. If Figure 1-9 This is the pore size distribution diagram of non-woven filter membrane.

[0045] 2. Assembly test of leukocyte filter

[0046] The modified filter membrane was cut using a slicer to prepare a leukocyte filter membrane with a diameter of 6 cm. The total number of filter membrane layers is 8, including 2 coarse filter membrane layers, 3 intermediate filter membrane layers, and 3 final filter membrane layers from top to bottom. The thickness of each filter membrane in the coarse filter layer and the intermediate layer is between 0.300-0.350 μm. The thickness of each final filter layer is 0.650-0.700 μm. The collected human blood was used for the leukocyte removal experiment, and 400 ml of blood was filtered each time.

[0047] 3. Blood filtration results

[0048] The blood cell content in the 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 the blood. However, when the blood is processed through a leukocyte filter, the leukocyte content may drop to very low levels. In this case, a traditional blood routine instrument may not provide sufficiently accurate measurement results. Therefore, we turned to the microscope counting method in "YY0329-2009 Disposable Leukocyte Removal Filter" to calculate the remaining leukocyte content.

[0049] The calculation formula of the number of white blood cells remaining in the leukocyte removal filter is:

[0050] Where:

[0051] LR——remaining white blood cell count, in pieces;

[0052] n——the average value of the white blood cell count of 3 tubes of samples, in pieces;

[0053] V – volume of blood components after filtration, in milliliters;

[0054] Vs - volume of blood sample for counting, in microliters;

[0055] 103——Conversion factor, 1mL=103μL

[0056] The formula for calculating the red blood cell recovery rate;

[0057] Where:

[0058] ER—erythrocyte recovery rate, %;

[0059] Rq——Red blood cell concentration of blood sample before filtration, reading value of blood cell counter;

[0060] Rh——Red blood cell concentration of filtered blood sample, blood cell counter reading;

[0061] Vq – volume of blood sample before filtration, in milliliters;

[0062] Vh – Volume of blood sample after filtration, in milliliters.

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

[0064] name Example 1 Example 2 Example 3 Domestic filter Blood volume before filtration (mL) 400 400 400 400 Filtered blood volume (mL) 378 381 380 371 <![CDATA[Original white blood cell count in whole blood (10 9 / L)]]> 5.52 5.62 5.47 5.98 <![CDATA[Number of leukocytes after filtration (10 6 cells / unit)]]> 0.87 0.73 0.82 0.91 Leukocyte removal rate (%) 99.984 99.987 99.985 99.985 <![CDATA[Original number of erythrocytes (10 12 / L)]]> 3.72 3.62 3.79 3.59 <![CDATA[Number of red blood cells after filtration (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 competing products, we found that these examples meet the requirements of the "YY0329-2009 Disposable Leukocyte Removal Filter" standard in terms of white blood cell removal rate and red blood cell recovery rate. Specifically, the white blood cell removal rate of the example is comparable to that of domestic filters. However, in terms of the two key indicators of blood recovery volume and red blood cell recovery rate, the performance of the example is better. The specific improvement is that the volume of blood after filtration has increased by 10 ml, and the red blood cell recovery rate has increased from 88.36% to 95.78%. These improvements not only reduce the waste of blood, but also have a positive impact on alleviating the current tight blood supply, the so-called "blood shortage."

[0066] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

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, use a constant temperature water bath to stir at 50-90°C for 60-300 minutes, and obtain a hydrophilic modifier solution after complete dissolution. Then, put a coarse filter layer non-woven fabric with a pore size of 12-20 μm into 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 3 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% of monomer and 90-99.5% of pure water to a three-necked flask, stir at 50-90°C for 60-300 minutes using a constant temperature water bath, and obtain a hydrophilic monomer solution after complete dissolution; then add 2-20% of initiator to the three-necked flask, stir using a magnetic stirrer, and heat at 50-80°C for 5-60 minutes during the stirring process to obtain an initiator solution; then place the middle layer non-woven fabric with a pore size of 8-14 μm in the hydrophilic monomer solution for 5-30 minutes, then place it in the initiator solution for 5-30 minutes, use an oven to cure at 70-100°C for half an hour, then wash with water 3 times, ultrasonically wash for 15 minutes, and dry to obtain a hydrophilic positively charged middle 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, 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, cured in an oven at 80-120°C for half an hour, washed with water for 3 times, ultrasonically washed for 15 minutes, and dried to obtain a hydrophilically modified final filter layer filter 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, characterized in that: 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 nonwoven fabric in step S1, the intermediate filter layer nonwoven fabric in step S2 and the final filter layer nonwoven fabric in step S3 are all selected from the group consisting of PBT, PET, PP, PA and PVC.

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