WO2020220157A1 - 一种全血过滤的方法及用于全血过滤的滤膜结构 - Google Patents

一种全血过滤的方法及用于全血过滤的滤膜结构 Download PDF

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WO2020220157A1
WO2020220157A1 PCT/CN2019/084755 CN2019084755W WO2020220157A1 WO 2020220157 A1 WO2020220157 A1 WO 2020220157A1 CN 2019084755 W CN2019084755 W CN 2019084755W WO 2020220157 A1 WO2020220157 A1 WO 2020220157A1
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filter membrane
membrane
hemagglutinin
filter
whole blood
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PCT/CN2019/084755
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English (en)
French (fr)
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许行尚
陈杰弗瑞
于沛
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南京岚煜生物科技有限公司
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Priority to SG11202108666YA priority Critical patent/SG11202108666YA/en
Priority to EP19927251.9A priority patent/EP3808434B1/en
Priority to US17/059,473 priority patent/US20210205524A1/en
Publication of WO2020220157A1 publication Critical patent/WO2020220157A1/zh

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D69/00—Semi-permeable membranes for separation processes or apparatus characterised by their form, structure or properties; Manufacturing processes specially adapted therefor
    • B01D69/12—Composite membranes; Ultra-thin membranes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
    • B01D61/58—Multistep processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D69/00—Semi-permeable membranes for separation processes or apparatus characterised by their form, structure or properties; Manufacturing processes specially adapted therefor
    • B01D69/02—Semi-permeable membranes for separation processes or apparatus characterised by their form, structure or properties; Manufacturing processes specially adapted therefor characterised by their properties
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D69/00—Semi-permeable membranes for separation processes or apparatus characterised by their form, structure or properties; Manufacturing processes specially adapted therefor
    • B01D69/14—Dynamic membranes
    • B01D69/141—Heterogeneous membranes, e.g. containing dispersed material; Mixed matrix membranes
    • B01D69/142—Heterogeneous membranes, e.g. containing dispersed material; Mixed matrix membranes with "carriers"
    • B01D69/144—Heterogeneous membranes, e.g. containing dispersed material; Mixed matrix membranes with "carriers" containing embedded or bound biomolecules
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D69/00—Semi-permeable membranes for separation processes or apparatus characterised by their form, structure or properties; Manufacturing processes specially adapted therefor
    • B01D69/14—Dynamic membranes
    • B01D69/141—Heterogeneous membranes, e.g. containing dispersed material; Mixed matrix membranes
    • B01D69/147—Heterogeneous membranes, e.g. containing dispersed material; Mixed matrix membranes containing embedded adsorbents
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/49—Blood
    • G01N33/491—Blood by separating the blood components
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/36—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits
    • A61M1/3621—Extra-corporeal blood circuits
    • A61M1/3627—Degassing devices; Buffer reservoirs; Drip chambers; Blood filters
    • A61M1/3633—Blood component filters, e.g. leukocyte filters
    • A61M1/3635—Constructional details
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2323/00—Details relating to membrane preparation
    • B01D2323/15—Use of additives
    • B01D2323/216—Surfactants
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2325/00—Details relating to properties of membranes
    • B01D2325/10—Catalysts being present on the surface of the membrane or in the pores
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2325/00—Details relating to properties of membranes
    • B01D2325/36—Hydrophilic membranes

Definitions

  • the invention belongs to the technical field of medical detection, and in particular relates to a method for whole blood filtration and a filter membrane structure for whole blood filtration.
  • the prior art generally uses plasma or serum samples obtained by centrifuging whole blood to determine the types and concentrations of blood components (such as metabolites, proteins, lipids, electrolytes, enzymes, antigens, and antibodies).
  • blood components such as metabolites, proteins, lipids, electrolytes, enzymes, antigens, and antibodies.
  • centrifugation is laborious and time-consuming, and the subsequent need to use a pipette to carefully remove the upper layer of serum or plasma can easily lead to remixing and the extraction volume is small.
  • centrifugation requires a centrifuge and electricity, it is not suitable for emergency situations and on-site inspections where a small amount of samples are quickly measured. Therefore, it is necessary to develop a device and method that can efficiently separate serum or plasma from whole blood.
  • Chinese patent document discloses a whole blood filtration and quantitative pipetting microfluidic chip; including a chip main body, and a whole blood separation mechanism, an anti-backflow microvalve, and a liquid quantitative mechanism provided on the chip main body , Propellant mechanism, flow-blocking microvalve and liquid outlet mechanism; among them: the whole blood separation mechanism includes a liquid inlet, a whole blood filter membrane and a collection unit arranged in sequence, which are used to filter and separate blood to obtain plasma, and under capillary action
  • the anti-backflow microvalve is used to transmit to the liquid quantitative mechanism;
  • the anti-backflow microvalve is used to prevent the plasma in the liquid quantitative mechanism from flowing back to the whole blood separation mechanism;
  • the flow-blocking microvalve is used to prevent the plasma from flowing out without external pressure;
  • the propellant mechanism It is used to push the quantitative plasma through the obstruction microvalve to the liquid outlet mechanism under pressure by the propellant; the whole blood filtration and quantitative pipetting microfluidic chip of the present invention can effectively separate blood
  • the disadvantage of this invention is that it cannot guarantee the blood filtration efficiency of the whole blood filter membrane, is prone to clogging, the plasma/serum filtration rate is low, and the application range is narrow, and it cannot guarantee the need for a large amount of plasma/serum use outside the microfluidic chip , The manufacturing cost of the microfluidic chip is high, and the process is complicated.
  • Chinese Patent Literature discloses a blood filter and a method for filtering blood.
  • the method includes the following steps: a. Providing a blood filter, the blood filter including a first side and A filter membrane on the second side and a receiving chamber defining a hollow space; and b. injecting a blood sample into the receiving chamber, wherein the volume of the hollow space is 3 to 20 times larger than the volume of the blood sample, by This increases the gas pressure in the receiving chamber so that the blood sample is filtered by the filter membrane, and the plasma or serum contained in the blood sample is forced to pass through the filter membrane.
  • the present invention also relates to a method for filtering blood to produce plasma or serum, the method comprising the following steps: a.
  • the blood filter comprising a filter having opposite first and second sides A membrane and a receiving chamber having a first volume; b. Inject a blood sample and gas into the syringe, the blood sample occupies the second volume of the syringe and the gas occupies the third volume in the syringe; c. Connecting the syringe to the blood filter so that they are in fluid communication with each other; and d. increasing the pressure in the syringe until the blood sample is received by the receiving chamber, so that the blood sample is received by the filter membrane Filtration; wherein the plasma or serum contained in the blood sample is forced to pass through the filter membrane; the sum of the first volume and the third volume is 3 to 20 times larger than the second volume .
  • the present invention also relates to a filter membrane having opposite first and second sides; the receiving chamber defines a hollow space for receiving a blood sample to be filtered, and the receiving chamber has at least one opening, The at least one opening is covered by the filter membrane, wherein the first side faces the receiving chamber, and the volume of the hollow space of the receiving chamber is 3 to 20 times larger than the volume of the blood sample to be filtered ;
  • the sampling chamber is arranged on the second side of the filter membrane.
  • the technical problem to be solved by the present invention is to provide a whole blood with high filtration efficiency, convenient operation, reliable quality, low cost, wide application range, and capable of effectively and stably separating plasma/serum from a very small amount of blood.
  • the filtering method achieves no leakage and hemolysis.
  • the technical solution adopted by the present invention is that the whole blood filtration method specifically includes the following steps:
  • the filter membrane structure is composed of at least two layers of filter membranes superimposed, and the filter membrane structure is subjected to hemagglutinin treatment, that is, hemagglutinin is added to the filter membrane structure, which can make the whole blood sample
  • hemagglutinin is added to the filter membrane structure, which can make the whole blood sample
  • the red blood cells are combined with the hemagglutinin in the filter membrane and are trapped in the filter membrane structure, while also trapping white blood cells and other impurities; thereby ensuring that the red blood cells are completely filtered and adsorbed in the filter membrane structure to ensure that the red blood cells in the blood are not filtered. It can effectively and stably separate plasma/serum from a very small amount of blood to achieve no leakage and hemolysis; hemagglutinin can be such as red blood cell antibodies.
  • a further improvement of the present invention is that the filter membrane has a porous structure; the pore size of the filter membrane superimposed in the filter membrane structure gradually decreases from top to bottom, and the area gradually becomes larger or equal.
  • the filter membrane structure is not less than two layers of filter membranes, and more layers of filter membranes can be superimposed; the filter membranes are all porous, and the uppermost layer is a loose porous structure, that is, the uppermost layer has the largest pore size, and the superimposed filter membrane in the filter membrane structure The pore size gradually decreases from top to bottom.
  • This arrangement facilitates layer-by-layer filtration, facilitates the passage of plasma or serum, and prevents blood cells and other impurities; the area of the superimposed filter membrane in the filter membrane structure can be equal from top to bottom Yes, it can also be gradually enlarged from top to bottom. This arrangement is conducive to filtering layer by layer, ensuring that the blood cells and other impurities leaked from the upper layer can continue to be filtered from the lower layer.
  • the filter membrane structure includes two layers of filter membranes, an upper filter membrane and a lower filter membrane, the upper filter membrane is a hemagglutinin filter membrane, and the lower filter membrane consists of at least one A water-based microporous membrane is superimposed; or the upper filter membrane is a hydrophilic filter membrane, and the lower filter membrane is a hemagglutinin filter; the hemagglutinin is evenly distributed in the hemagglutinin filter.
  • the hemagglutinin can be dispersed on the upper filter membrane or the lower filter membrane. It is only necessary to ensure that the hemagglutinin is in the filter membrane structure, so that the red blood cells can be completely filtered and adsorbed in the filter membrane structure to ensure that the red blood cells in the blood are not filtered Over.
  • the upper filter membrane is a hemagglutinin filter membrane
  • the lower filter membrane is a filter membrane with a number of evenly distributed filter holes and different pore sizes, or the lower filter membrane is composed of a first lower layer
  • the membrane and the second lower layer film are sequentially stacked up and down, and the pore size of the first lower layer film is larger than the pore size of the second lower layer film.
  • the preferred solution is to add hemagglutinin to the upper filter membrane, so that the red blood cells can be agglutinated in the upper filter membrane, while the serum or plasma enters the lower filter membrane.
  • the upper filter membrane is glass fiber filter paper or nitrocellulose membrane or polysulfone membrane
  • the lower filter membrane is a hydrophilic microporous membrane, including glass fiber filter paper or nitrocellulose membrane or Polysulfone membrane or cellulose acetate membrane
  • the step (2) adopts upper pressure or lower suction to accelerate the filtration speed during the filtration.
  • the formed filter membrane structure can withstand a pressure of 1-30 MPa, which is convenient for active air pressure to promote blood filtration.
  • a further improvement of the present invention is that the hemagglutinin filter membrane is obtained by subjecting the filter membrane to hemagglutinin treatment, and the preparation process is as follows:
  • step 2) Put the filter membrane into the hemagglutinin buffer solution containing hemagglutinin in step 1), then dry it at room temperature, and then place it in an oven at 37°C to 55°C for more than 2 hours; or The filter membrane moistened in the hemagglutinin buffer is vacuum-dried or freeze-dried.
  • the hemagglutinin buffer is PB or Tris-HCl or CB; the concentration of the hemagglutinin buffer is 5mM-1M; the hemagglutinin added to the hemagglutinin filter
  • the weight of the vegetarian is not less than 5ng.
  • PB is sodium phosphate buffer (NaH 2 PO 4 &Na 2 HPO 4 ) and potassium phosphate buffer (K 2 HPO 4 &KH 2 PO 4 );
  • Tris-HCl is tris(hydroxymethyl) aminomethane;
  • CB buffer is Carbonate buffer.
  • a further improvement of the present invention is that the filter membrane needs to be treated with a buffer solution before use, and the buffer solution is any buffer solution that dissolves BSA, amino acids, and contains surface active agent components; the processing method is: After the filter membrane is wetted in the buffer solution, it is dried at room temperature and dried for more than 2 hours, ready for use. Each layer of the filter membrane is treated with a buffer to avoid protein adsorption in serum or plasma, and the filtered serum or plasma can be used for other purposes, such as the detection of certain substances.
  • the pore size of the upper filter membrane is not less than 0.45 ⁇ m, and the pore size of the lower filter membrane is 0.2 ⁇ m to 4 ⁇ m.
  • the upper filter membrane has a pore size of 1 ⁇ m to 5 ⁇ m; the upper filter membrane has a thickness of 0.5 to 20 mm, and the lower filter membrane has a thickness of 0.05 to 2 mm; the hemagglutinin filter The weight of hemagglutinin added to the membrane is 20 ng to 100 ng.
  • Both the upper filter membrane and the lower filter membrane can be a single layer, or can be stacked in multiple layers to achieve the required thickness.
  • the technical problem to be solved by the present invention is to provide a whole blood filtration membrane structure that can effectively and stably separate plasma/serum from a very small amount of blood.
  • the filter membrane structure for whole blood filtration is composed of at least two layers of filter membranes sequentially stacked from top to bottom; the filter membrane has a porous structure; the filter membrane The pore size of the stacked filter membranes gradually decreases from top to bottom, and the area gradually becomes larger or equal from top to bottom.
  • the filter membrane structure is not less than two layers of filter membranes, and more layers of filter membranes can be superimposed; the filter membranes are all porous, and the uppermost layer is a loose porous structure, that is, the uppermost layer has the largest pore size, and the superimposed filter membrane in the filter membrane structure The pore size gradually decreases from top to bottom.
  • This arrangement facilitates layer-by-layer filtration, facilitates the passage of plasma or serum, and prevents blood cells and other impurities; the area of the superimposed filter membrane in the filter membrane structure can be equal from top to bottom Yes, it can also be gradually enlarged from top to bottom. This arrangement is conducive to filtering layer by layer, ensuring that the blood cells and other impurities leaked from the upper layer can continue to be filtered from the lower layer.
  • the filter membrane structure includes two layers of filter membranes, an upper filter membrane and a lower filter membrane, the upper filter membrane is a hemagglutinin filter membrane, and the lower filter membrane consists of at least one layer of hydrophilic Microporous membrane superimposed composition; or the upper filter membrane is a hydrophilic filter membrane, the lower filter membrane is a hemagglutinin filter membrane; the hemagglutinin is evenly distributed in the hemagglutinin filter.
  • the hemagglutinin can be dispersed on the upper filter membrane or the lower filter membrane. It is only necessary to ensure that the hemagglutinin is in the filter membrane structure, so that the red blood cells can be completely filtered and adsorbed in the filter membrane structure to ensure that the red blood cells in the blood are not filtered Over.
  • the present invention is further improved in that the upper filter membrane is a hemagglutinin filter membrane; the lower filter membrane is a filter membrane with a number of evenly distributed filter holes and different pore sizes, or the lower filter membrane consists of a first lower membrane and The second lower film is formed by stacking up and down sequentially, and the pore size of the first lower film is larger than the pore size of the second lower film.
  • the pore size of the upper filter membrane is not less than 0.45 ⁇ m, and the pore size of the lower filter membrane is 0.2 ⁇ m to 4 ⁇ m.
  • the upper filter membrane has a pore size of 1 ⁇ m to 5 ⁇ m; the upper filter membrane has a thickness of 0.5 to 20 mm, and the lower filter membrane has a thickness of 0.05 to 2 mm; the hemagglutinin filter The weight of hemagglutinin added to the membrane is 20 ng to 100 ng.
  • the present invention has the following beneficial effects:
  • Coagulation/anti-coagulation chemicals or antibodies/antigens are added to the filter layer to agglutinate blood cells and contained in the filter layer. Only serum or plasma can pass through; avoid improper pressure control and hemolysis that affects the test results accuracy;
  • This method can guarantee blood filtration regardless of the content of blood cells in the whole blood sample; according to the size of hematocrit and the volume of whole blood, the filtration output of plasma/serum can be predicted, on the contrary, quantitative collection can be realized Plasma/serum; a large amount of plasma/serum can be collected by continuously adding whole blood;
  • This method can filter vertically or laterally and has a wide range of applications.
  • Figure 1 is a flow chart of the method of whole blood filtration of the present invention
  • FIG. 2 is a longitudinal cross-sectional screenshot of the filter membrane structure for whole blood filtration in Example 31 and Example 33 of the present invention
  • Fig. 3 is a longitudinal cross-sectional screenshot of the filter membrane structure for whole blood filtration of embodiment 32 of the present invention
  • 1-upper filter membrane 2-lower filter membrane; 201-first lower membrane; 202-second lower membrane.
  • the filter membrane structure includes two layers of filter membranes, the upper filter membrane 1 and the lower filter membrane 2, respectively.
  • the upper filter membrane 1 is a glass fiber paper with a thickness of 0.5 mm and a pore size of 5 ⁇ m.
  • the RBC antibody red blood cell antibody is A type of hemagglutinin
  • PB buffer a glass fiber paper with a thickness of 0.5 mm and a pore size of 5 ⁇ m.
  • the RBC antibody red blood cell antibody is A type of hemagglutinin
  • the upper filter membrane 1 is processed to make the RBC antibody content in the upper filter membrane 1 100ng; then placed in an oven at 37°C ⁇ 55°C to dry for more than 2h ;
  • the lower filter membrane 2 is made of glass fiber paper with a thickness of 1mm, treated with 1% BSA dissolved in PB buffer; dried at room temperature and dried for more than 2 hours;
  • the whole blood filtration method specifically includes the following steps:
  • the filtration effect can complete the filtration of normal 200 ⁇ L whole blood serum within 30s, and the filtered serum can reach more than 60 ⁇ L.
  • Examples 2 to 4 all use the filter membrane structure of Example 1 above, and the method of whole blood filtration is the same; the difference from Example 1 is the thickness of the upper filter membrane 1 and the lower filter membrane 2, the filtration time and the collected Serum levels are shown in Table 1.
  • Example 1 0.5 1 27 65 no Example 2 0.25 1 17 70 Have blood cells
  • Example 3 0.5 2 65 50 no Example 4 0.25 2 60 50 no
  • Example 1 has the best effect.
  • Examples 5 to 7 all use the membrane structure of Example 1 above, and the method of whole blood filtration is also the same; the difference from Example 1 is that the pressure applied during the filtration in step (2) is different, and the filtration conditions are as follows Table 2 shows.
  • Example 1 has the best effect.
  • Examples 8-15 all use the whole blood filtration method of Example 1 above, and the parameters in the filtration are the same, and the filter membrane structure also uses the filter membrane structure of Example 1 above.
  • the difference from Example 1 is that the filter membrane structure
  • the selected materials of the upper filter membrane 1 and the lower filter membrane 2 are different; the filtration conditions are shown in Table 3 below.
  • Example 1 has the best effect.
  • Example 16 Ferric chloride is used as the hemagglutinin in this example; taking a 200 ⁇ L whole blood sample as an example, when the whole blood sample contains 50% serum, this structure can filter out no less than 50 ⁇ L volume Serum, the amount of serum filtered is more than 50%; the filter membrane structure includes two layers of filter membranes, the upper filter membrane 1 and the lower filter membrane 2, respectively.
  • the upper filter membrane 1 is a glass fiber paper with a thickness of 0.5 mm and a pore size of 5 ⁇ m.
  • a ferric chloride solution with a concentration of 50 mM treats the upper filter membrane 1 so that ferric chloride is evenly distributed in the upper filter membrane 1, and the iron chloride content is 100 ng;
  • the lower filter membrane 2 is made of glass fiber paper with a thickness of 1mm, treated with 1% BSA dissolved in PB buffer; dried at room temperature and dried for more than 2 hours;
  • the whole blood filtration method specifically includes the following steps:
  • the filtration effect can complete the filtration of normal 200 ⁇ L whole blood serum within 60s, and the filtered serum reaches more than 60 ⁇ L.
  • Examples 17 to 19 adopt the filter membrane structure of Example 16 and the same treatment method for the filter membrane structure, and the same method of whole blood filtration; the difference from Example 16 is the upper filter membrane 1 and the lower filter membrane The thickness of 2, its filtration time and the amount of serum collected are shown in Table 4.
  • Example 16 has the best effect.
  • Examples 20-22 all use the filter membrane structure of the above-mentioned Example 16 and the same treatment method for the filter membrane structure, and the same method of whole blood filtration; the difference from Example 16 is the filtration in step (2) When the pressure applied to the upper part is different, the filtering conditions are shown in Table 5 below.
  • Example 16 has the best effect.
  • Examples 23-30 all use the whole blood filtration method of Example 16 above, and the parameters in the filtration are the same, and the filter membrane structure also uses the filter membrane structure of Example 16 above and the same processing method for the filter membrane structure.
  • the difference in embodiment 16 is that the materials selected for the upper filter membrane 1 and the lower filter membrane 2 in the filter membrane structure are different; the filtration conditions are shown in Table 6 below.
  • Example 16 has the best effect.
  • the filter membrane structure for whole blood filtration consists of two layers of filter membranes stacked from top to bottom in sequence; the filter membrane is a porous structure; The pore size of the membrane gradually becomes smaller from top to bottom, and the area gradually becomes larger from top to bottom; the filter membrane structure includes two layers of filter membranes, an upper filter membrane 1 and a lower filter membrane 2, respectively, the upper filter membrane 1 It is a hemagglutinin filter membrane, the lower filter membrane 2 is a filter membrane with a number of evenly distributed filter holes and different pore sizes; the upper filter membrane 1 has a pore size of 1 ⁇ m, and the lower filter membrane 2 has a pore size of 0.2-0.5 ⁇ m; the thickness of the upper filter membrane 1 is 0.5mm, the thickness of the lower filter membrane 2 is 1mm; the hemagglutinin filter membrane is a filter membrane after hemagglutinin treatment, in which hemagglutinin is evenly distributed The weight of the hemagglutinin is 100
  • Embodiment 32 As shown in FIG. 3, the difference from embodiment 31 is that the lower filter membrane 2 is composed of a first lower film 201 and a second lower film 202 stacked one above the other in sequence, and the first lower film The pore size of 201 is larger than the pore size of the second lower membrane 202.
  • the filter membrane structure for whole blood filtration is composed of two layers of filter membranes sequentially stacked from top to bottom;
  • the filter membrane is a porous structure;
  • the pore size of the stacked filter membranes in the filter membrane structure is from top to bottom The pore size gradually becomes smaller and the area gradually becomes larger from top to bottom;
  • the filter membrane structure includes two layers of filter membranes, an upper filter membrane 1 and a lower filter membrane 2, respectively, and the upper filter membrane 1 is a hemagglutinin filter membrane
  • the lower filter membrane 2 is composed of a first lower film 201 and a second lower film 202 stacked one above the other in sequence, and the pore diameter of the first lower film 201 is larger than the pore diameter of the second lower film 202.
  • the pore size of the first lower layer membrane 201 is 0.5 ⁇ m, the pore size of the second lower layer membrane 202 is 0.2 ⁇ m; the thickness of the upper layer filter membrane 1 is 0.5 mm, and the total thickness of the lower layer filter membrane 2 is 1 mm , Wherein the thickness of the first lower film 201 is 0.5 mm, and the thickness of the second lower film 202 is 0.5 mm.
  • Example 33 As shown in Figure 2, the difference from Example 31 is that the upper filter membrane 1 is a hydrophilic filter membrane, and the lower filter membrane 2 is a hemagglutinin filter membrane; specifically: The filter membrane structure of blood filtration is composed of two layers of filter membranes sequentially stacked from top to bottom; the filter membrane is a porous structure; the pore size of the filter membrane superimposed in the filter membrane structure gradually decreases from top to bottom, and the area Gradually increase from top to bottom; the filter membrane structure includes two layers of filter membranes, an upper filter membrane 1 and a lower filter membrane 2, the upper filter membrane 1 is a hydrophilic filter membrane, and the lower filter membrane 2 is red blood cell Lectin filter membrane; the pore size of the upper filter membrane 1 is 1 ⁇ m, the pore size of the lower filter membrane 2 is 0.2 to 0.5 ⁇ m; the thickness of the upper filter membrane 1 is 0.5 mm, and the thickness of the lower filter membrane 2
  • the hemagglutinin filter membrane is a filter membrane treated with
  • the filter membrane structure is not less than two layers of filter membranes, and more layers of filter membranes can be superimposed; the filter membranes are all porous, and the uppermost layer is a loose porous structure, that is, the uppermost layer has the largest pore size, and the superimposed filter membrane in the filter membrane structure
  • the pore size gradually decreases from top to bottom.
  • This arrangement facilitates layer-by-layer filtration, facilitates the passage of plasma or serum, and prevents blood cells and other impurities; the area of the superimposed filter membrane in the filter membrane structure can be equal from top to bottom Yes, it can also gradually increase from top to bottom.
  • This arrangement is beneficial to filtering layer by layer, ensuring that the blood cells and other impurities leaked from the upper layer can continue to be filtered from the lower layer.

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Abstract

一种全血过滤的方法及用于全血过滤的滤膜结构，具体包括以下步骤：(1)选择由至少两层过滤膜依次从上至下叠加构成滤膜结构，且将所述滤膜结构进行红细胞凝集素处理，待用；(2)将全血样本加入所述滤膜结构进行过滤；(3)收集过滤后的血清或血浆。滤膜结构为至少两层过滤膜从上至下叠加构成，且叠加的过滤膜从上至下的孔径逐渐变小，面积逐渐变大或相等。通过由至少两层过滤膜叠加构成的滤膜结构，且将所述滤膜结构进行红细胞凝集素处理，使全血样本中的红细胞与滤膜中红细胞凝集素相结合，被截留在滤膜结构中，从而以保证红细胞完全过滤吸附在滤膜结构中，从而实现能从非常少量的血液中有效稳定地分离出血浆/血清。

Description

一种全血过滤的方法及用于全血过滤的滤膜结构 技术领域
本发明属于医疗检测技术领域，尤其是涉及一种全血过滤的方法及用于全血过滤的滤膜结构。
背景技术
现有技术通常利用通过离心全血得到的血浆或血清样品来测定血液成份(例如代谢物、蛋白质、脂质、电解质、酶类、抗原和抗体)的种类和浓度。然而，离心费力又耗时，且后续需要利用移液器小心移取出上层血清或血浆，容易导致重新混合，提取量偏少。特别是，由于离心需要离心机和电力，因此不适于迅速测定少量样品的紧急情况和现场检验。因此，研究出一种能高效从全血中分离血清或血浆的装置和方法很有必要。
中国专利文献(申请号为：201610200920.7)公开了一种全血过滤及定量移取微流控芯片；包括芯片主体、以及设置在芯片主体上的全血分离机构、防倒流微阀、液体定量机构、推进液机构、阻流微阀和出液机构；其中：全血分离机构包括依次设置的进液口、全血滤膜和收集单元，用于将血液过滤分离得到血浆、并在毛细作用下经防倒流微阀传送至液体定量机构；防倒流微阀用于阻止液体定量机构中的血浆逆流至全血分离机构；阻流微阀用于阻止血浆在无外界压力情况下流出；推进液机构用于将推进液在压力作用下将定量血浆经阻流微阀推送至出液机构；本发明的全血过滤及定量移取微流控芯片，有效分离血细胞和血浆并定量移取血浆、适合与其他类型芯片组合使用。该发明的缺点在于不能保证全血滤膜的滤血效率，容易发生堵塞，血浆/血清滤出率低，而且应用范围较窄，不能保证微流控芯片之外的大量血浆/血清使用的需求，微流控芯片的制造成本高，工艺复杂。
中国专利文献(申请号为：201080026906.0)公开了一种血液过滤器以及过滤血液的方法，所述方法包括以下步骤：a.提供血液过滤器，所述血液过滤器包括具有相反的第一侧和第二侧的滤膜以及限定中空空间的接收室；和b.将血液样品注入到所述接收室中，其中，所述中空空间的容积比所述血液样品的体积大3至20倍，由此增加所述接收室内的气体压力，使得所述血液样品被所述滤膜过 滤，并且包含在所述血液样品中的所述血浆或血清被迫通过所述滤膜。本发明还涉及一种用于过滤血液以产生血浆或血清的方法，所述方法包括以下步骤：a.提供血液过滤器，所述血液过滤器包括具有相反的第一侧和第二侧的滤膜以及具有第一容积的接收室；b.将血液样品和气体注入到注射器中，所述血液样品占用所述注射器的第二体积并且所述气体占用所述注射器中的第三体积；c.将所述注射器连接到所述血液过滤器以使得它们彼此流体连通；以及d.增加所述注射器内的压力直到所述血液样品被所述接收室接收，使得所述血液样品被所述滤膜过滤；其中，被包含在所述血液样品中的所述血浆或血清被迫通过所述滤膜；所述第一容积和所述第三体积之和比所述第二体积大3至20倍。本发明还涉及一种滤膜，所述滤膜具有相反的第一侧和第二侧；所述接收室限定用于接收待过滤的血液样品的中空空间，所述接收室具有至少一个开口，所述至少一个开口被所述滤膜覆盖，其中，所述第一侧面向所述接收室，并且所述接收室的所述中空空间的容积比待过滤的血液样品的体积大3至20倍；所述采样室被布置在所述滤膜的所述第二侧。该发明的缺点是需要平衡使用者的施加压力，减少红细胞溶血现象的发生，使用者若使用不当，施加压力过大易造成溶血现象，影响血浆成份进而可能影响检测项目的结果准确性。而且利用具有柱塞的注射器加样，易造成血细胞过滤分布不均，注射器对应滤膜部分易发生堵塞，影响过滤效率。
因此，有必要研发出一种全血过滤效率高，方便操作，质量可靠，成本低，适用范围广泛的全血过滤方法及用于全血过滤的滤膜结构。
发明内容
本发明要解决的技术问题是提供一种全血过滤效率高，方便操作，质量可靠，成本低，适用范围广泛的，且能从非常少量的血液中有效稳定地分离出血浆/血清的全血过滤的方法，实现无漏出和溶血作用。
为解决上述技术问题，本发明采用的技术方案是，该全血过滤的方法，具体包括以下步骤：
(1)选择由至少两层过滤膜依次从上至下叠加构成滤膜结构，且将所述滤膜结构进行红细胞凝集素处理，待用；
(2)将全血样本加入所述滤膜结构进行过滤；
(3)设置收集装置收集过滤后的血清或血浆。
采用上述技术方案，通过由至少两层过滤膜叠加构成的滤膜结构，且将所述滤膜结构进行红细胞凝集素处理，即在滤膜结构中添加红细胞凝集素，能使全血样本中的红细胞与过滤膜中红细胞凝集素相结合，被截留在过滤膜结构中，同时还可以截留白细胞与其他杂质；从而保证红细胞完全过滤吸附在滤膜结构中，保证血液中红细胞不滤过，从而实现能从非常少量的血液中有效稳定地分离出血浆/血清，实现无漏出和溶血作用；红细胞凝集素可以是诸如红细胞抗体等。
本发明的进一步改进在于，所述过滤膜为多孔结构；所述滤膜结构中叠加的过滤膜从上至下的孔径逐渐变小，面积逐渐变大或相等。滤膜结构不少于两层过滤膜，也可以叠加更多层过滤膜；过滤膜均为多孔结构，且最上层为疏松多孔结构，即最上层的孔径最大，滤膜结构中叠加的过滤膜从上往下孔径依次逐渐减小，这样的设置便于层层过滤，便于血浆或血清通过，而使血细胞与其他杂质阻留；滤膜结构中叠加的过滤膜从上至下的面积可以是相等的，也可以是从上至下逐渐变大，这样的设置有利于层层过滤，保证上层漏掉的血细胞与其他杂质可以由下层继续过滤。
本发明的进一步改进在于，所述滤膜结构包括两层过滤膜，分别为上层过滤膜和下层过滤膜，所述上层过滤膜为红细胞凝集素滤膜，所述下层过滤膜由至少一层亲水性微孔膜叠加组成；或所述上层过滤膜为亲水过滤膜，所述下层过滤膜为红细胞凝集素滤膜；所述红细胞凝集素滤膜中均匀分布有红细胞凝集素。红细胞凝集素可以分散在上层过滤膜，也可以分散在下层过滤膜，只需要保证红细胞凝集素在滤膜结构中，这样便能保证红细胞完全过滤吸附在滤膜结构中，保证血液中红细胞不滤过。
作为本发明的优选技术方案，所述上层过滤膜为红细胞凝集素滤膜；所述下层过滤膜为若干滤孔均匀分布且孔径大小不同的过滤膜，或所述下层过滤膜由第一下层膜和第二下层膜依次上下叠加组成，所述第一下层膜的孔径大于所述第二下层膜的孔径。优选的方案是将红细胞凝集素添加在上层过滤膜中，这样可以使红细胞凝集在上层过滤膜中，而血清或血浆进入下层过滤膜。
作为本发明的优选技术方案，所述上层过滤膜为玻璃纤维滤纸或硝酸纤维素膜或聚砜膜；所述下层过滤膜为亲水性微孔膜，包括玻璃纤维滤纸或硝酸纤维素膜或聚砜膜或乙酸纤维素膜；所述步骤(2)过滤时采用上部加压或下部抽吸加 快过滤速度。所形成的滤膜结构可以承受1～30MPa的压强，便于主动式气压推动血液滤过。
本发明的进一步改进在于，所述红细胞凝集素滤膜是将过滤膜进行红细胞凝集素处理得到的，其制备过程为：
1)将红细胞凝集素放入红细胞凝集素缓冲液中稀释；
2)将所述过滤膜放入步骤1)的含有红细胞凝集素的红细胞凝集素缓冲液中润湿，然后在室温晾干，再放置于37℃～55℃温度烘箱中烘干2h以上；或将在红细胞凝集素缓冲液中润湿后的所述过滤膜进行真空抽干或冻干处理。
作为本发明的优选技术方案，所述红细胞凝集素缓冲液为PB或Tris-HCl或CB；所述红细胞凝集素缓冲液的浓度为5mM～1M；所述红细胞凝集素滤膜中添加的红细胞凝集素的重量不少于5ng。其中PB为磷酸钠缓冲液(NaH 2PO 4&Na 2HPO 4)和磷酸钾缓冲液(K 2HPO 4&KH 2PO 4)；Tris-HCl为三(羟甲基)氨基甲烷；CB缓冲液为碳酸盐缓冲液。
本发明的进一步改进在于，所述过滤膜在使用前需采用缓冲液进行处理，所述缓冲液为溶解BSA、氨基酸、含有表面活动性剂成分的任一种缓冲液；处理方法为：将所述过滤膜在缓冲液中润湿后，在室温晾干且干燥2h以上，待用。将各层过滤膜都用缓冲液处理，可以避免血清或血浆中的蛋白吸附，可以使过滤后的血清或血浆用于其他用途，例如其中某些物质检测等。
作为本发明的优选技术方案，所述上层过滤膜的孔径不小于0.45μm，所述下层过滤膜的孔径为0.2μm～4μm。
作为本发明的优选技术方案，所述上层过滤膜的孔径为1μm～5μm；所述上层过滤膜的厚度为0.5～20mm，所述下层过滤膜的厚度为0.05～2mm；所述红细胞凝集素滤膜中添加的红细胞凝集素的重量为20ng～100ng。所述上层过滤膜和所述下层过滤膜均可以用单层，也可以多层叠加的方式达到所需的厚度。
本发明还要解决的技术问题是提供一种能从非常少量的血液中有效稳定地分离出血浆/血清的全血过滤的滤膜结构。
为解决上述技术问题，本发明采用的技术方案是，该用于全血过滤的滤膜结构，由至少两层过滤膜依次从上至下叠加构成；所述过滤膜为多孔结构；该滤膜结构中叠加的过滤膜的孔径从上至下孔径逐渐变小，面积从上至下逐渐变大或相 等。滤膜结构不少于两层过滤膜，也可以叠加更多层过滤膜；过滤膜均为多孔结构，且最上层为疏松多孔结构，即最上层的孔径最大，滤膜结构中叠加的过滤膜从上往下孔径依次逐渐减小，这样的设置便于层层过滤，便于血浆或血清通过，而使血细胞与其他杂质阻留；滤膜结构中叠加的过滤膜从上至下的面积可以是相等的，也可以是从上至下逐渐变大，这样的设置有利于层层过滤，保证上层漏掉的血细胞与其他杂质可以由下层继续过滤。
本发明进一步改进在于，该滤膜结构包括两层过滤膜，分别为上层过滤膜和下层过滤膜，所述上层过滤膜为红细胞凝集素滤膜，所述下层过滤膜由至少一层亲水性微孔膜叠加组成；或所述上层过滤膜为亲水过滤膜，所述下层过滤膜为红细胞凝集素滤膜；所述红细胞凝集素滤膜中均匀分布有红细胞凝集素。红细胞凝集素可以分散在上层过滤膜，也可以分散在下层过滤膜，只需要保证红细胞凝集素在滤膜结构中，这样便能保证红细胞完全过滤吸附在滤膜结构中，保证血液中红细胞不滤过。
本发明进一步改进在于，所述上层过滤膜为红细胞凝集素滤膜；所述下层过滤膜为若干滤孔均匀分布且孔径大小不同的过滤膜，或所述下层过滤膜由第一下层膜和第二下层膜依次上下叠加组成，所述第一下层膜的孔径大于所述第二下层膜的孔径。
作为本发明的优选技术方案，所述上层过滤膜的孔径不小于0.45μm，所述下层过滤膜的孔径为0.2μm～4μm。
作为本发明的优选技术方案，所述上层过滤膜的孔径为1μm～5μm；所述上层过滤膜的厚度为0.5～20mm，所述下层过滤膜的厚度为0.05～2mm；所述红细胞凝集素滤膜中添加的红细胞凝集素的重量为20ng～100ng。
与现有技术相比，本发明具有的有益效果为：
(1)通过由至少两层过滤膜叠加构成的滤膜结构，实现了通过物理结构把全血中的血清或血浆滤出，吸附红细胞等物质；
(2)在滤层中添加了凝血/抗凝血的化学物质或抗体/抗原，可以使血细胞凝集，容载于滤层中，只有血清或血浆通过；避免压力控制不当发生溶血而影响检测结果准确性；
(3)该方法不管全血样本中的血细胞含量高低，都能保证滤血；根据血细 胞比容的大小和全血的容积，可以预测血浆/血清的过滤产出量，相反地可以实现定量收集血浆/血清；通过不断加入全血可以实现大量血浆/血清的收集；
(4)该方法能够垂直或侧向滤过，应用范围广。
附图说明
下面结合附图和本发明的实施方式进一步详细说明：
图1是本发明全血过滤的方法的流程图；
图2是本发明的实施例31和实施例33的用于全血过滤的滤膜结构的纵向剖面截图；
图3是本发明的实施例32的用于全血过滤的滤膜结构的纵向剖面截图；
其中1-上层过滤膜；2-下层过滤膜；201-第一下层膜；202-第二下层膜。
具体实施方式
实施例1：滤膜结构包括两层过滤膜，分别为上层过滤膜1和下层过滤膜2，上层过滤膜1的厚度为0.5mm的玻璃纤维纸，孔径5μm，用RBC抗体(红细胞抗体，为红细胞凝集素的一种)溶解到PB缓冲液，然后对上层过滤膜1进行处理，使上层过滤膜1中的RBC抗体含量为100ng；再放置于37℃～55℃温度烘箱中烘干2h以上；
下层过滤膜2采用1mm厚度的玻璃纤维纸，用PB缓冲液溶解1％的BSA处理；室温晾干并干燥2h以上；
如图1所示，该全血过滤的方法，具体包括以下步骤：
(1)选择上述滤膜结构；
(2)将全血样本加入所述滤膜结构进行过滤；过滤时采用上部加压过滤速度；
(3)设置收集装置收集过滤后的血清或血浆。
在主动加压20MPa过滤的情况下，过滤效果可以在30s内完成正常200μL全血中血清的滤过，并且过滤血清达到60μL以上。
实施例2～4均采用上述实施例1的滤膜结构，且全血过滤的方法也相同；与实施例1不同的是上层过滤膜1和下层过滤膜2的厚度，其过滤时间及收集的血清量如下表1。
表1 实施例1～4的全血过滤的结果
实施例 上层过滤膜厚度 下层过滤膜 平均过滤时 滤出血清体 有无血细胞
  (mm) 厚度(mm) 间(s) 积(μL) 滤过
实施例1 0.5 1 27 65 无
实施例2 0.25 1 17 70 有血细胞
实施例3 0.5 2 65 50 无
实施例4 0.25 2 60 50 无
从表1中可以看出，实施例1的效果最好。
实施例5～7均采用上述实施例1的滤膜结构，且全血过滤的方法也相同；与实施例1不同的是步骤(2)中的过滤时所加的压强不同，其过滤情况如下表2所示。
表2 实施例1、5～7的全血过滤的结果
Figure PCTCN2019084755-appb-000001
从表2中可以看出，实施例1的效果最好。
实施例8～15均采用上述实施例1的全血过滤方法，且过滤中的参数一致，滤膜结构也均采用上述实施例1的滤膜结构，与实施例1不同的是滤膜结构中的上层过滤膜1与下层过滤膜2的所选用的材料不同；其过滤情况如下表3所示。
表3 实施例1、8～15的全血过滤的结果
Figure PCTCN2019084755-appb-000002
从表3中可以看出，实施例1的效果最好。
实施例16：本实施例中的红细胞凝集素选用氯化铁；以200μL全血样本为例，当全血样本中含有50％比例的血清时，采用该结构可以滤出不少于50μL体积的血清，滤过血清量在50％以上；滤膜结构包括两层过滤膜，分别为上层过滤 膜1和下层过滤膜2，上层过滤膜1的厚度为0.5mm的玻璃纤维纸，孔径5μm，用浓度50mM的氯化铁溶液对上层过滤膜1进行处理，使上层过滤膜1中均匀分布有氯化铁，且氯化铁含量为100ng；
下层过滤膜2采用1mm厚度的玻璃纤维纸，用PB缓冲液溶解1％的BSA处理；室温晾干并干燥2h以上；
如图1所示，该全血过滤的方法，具体包括以下步骤：
(1)选择上述滤膜结构；
(2)将全血样本加入所述滤膜结构进行过滤；过滤时采用上部加压过滤速度；
(3)设置收集装置收集过滤后的血清或血浆。
在主动加压20MPa过滤的情况下，过滤效果可以在60s内完成正常200μL全血中血清的滤过，并且过滤血清达到60μL以上。
实施例17～19均采用上述实施例16的滤膜结构以及相同的对滤膜结构的处理方法，和相同的全血过滤的方法；与实施例16不同的是上层过滤膜1和下层过滤膜2的厚度，其过滤时间及收集的血清量如下表4。
表4 实施例16～19的全血过滤的结果
Figure PCTCN2019084755-appb-000003
从表4中可以看出，实施例16的效果最好。
实施例20～22均均采用上述实施例16的滤膜结构以及相同的对滤膜结构的处理方法，和相同的全血过滤的方法；与实施例16不同的是步骤(2)中的过滤时上部所加的压强不同，其过滤情况如下表5所示。
表5 实施例16、20～22的全血过滤的结果
Figure PCTCN2019084755-appb-000004
从表5中可以看出，实施例16的效果最好。
实施例23～30均采用上述实施例16的全血过滤方法，且过滤中的参数一致，滤膜结构也均采用上述实施例16的滤膜结构以及相同的对滤膜结构的处理方法，与实施例16不同的是滤膜结构中的上层过滤膜1与下层过滤膜2的所选用的材料不同；其过滤情况如下表6所示。
表6 实施例16、23～30的全血过滤的结果
Figure PCTCN2019084755-appb-000005
从表6中可以看出，实施例16的效果最好。
实施例31：如图2所示，该用于全血过滤的滤膜结构，由两层过滤膜依次从上至下叠加构成；所述过滤膜为多孔结构；该滤膜结构中叠加的过滤膜的孔径从上至下孔径逐渐变小，面积从上至下逐渐变大；所述滤膜结构包括的两层过滤膜分别为上层过滤膜1和下层过滤膜2，所述上层过滤膜1为红细胞凝集素滤膜，所述下层过滤膜2为若干滤孔均匀分布且孔径大小不同的过滤膜；所述上层过滤膜1的孔径为1μm，所述下层过滤膜2的孔径为0.2～0.5μm；所述上层过滤膜1的厚度为0.5mm，所述下层过滤膜2的厚度为1mm；所述红细胞凝集素滤膜为经过红细胞凝集素处理后的过滤膜，其中均匀分布有红细胞凝集素，所述红细胞凝集素的重量为100ng。
实施例32：如图3所示，与实施例31不同之处在于，所述下层过滤膜2由第一下层膜201和第二下层膜202依次上下叠加组成，所述第一下层膜201的孔径大于所述第二下层膜202的孔径。具体地：该用于全血过滤的滤膜结构，由两层过滤膜依次从上至下叠加构成；所述过滤膜为多孔结构；该滤膜结构中叠加的过滤膜的孔径从上至下孔径逐渐变小，面积从上至下逐渐变大；所述滤膜结构包括的两层过滤膜分别为上层过滤膜1和下层过滤膜2，所述上层过滤膜1为红细 胞凝集素滤膜，所述下层过滤膜2由第一下层膜201和第二下层膜202依次上下叠加组成，所述第一下层膜201的孔径大于所述第二下层膜202的孔径。所述第一下层膜201的孔径为0.5μm，所述第二下层膜202的孔径为0.2μm；所述上层过滤膜1的厚度为0.5mm，所述下层过滤膜2的总厚度为1mm，其中第一下层膜201的厚度为0.5mm，第二下层膜202的厚度为0.5mm。
实施例33：如图2所示，与实施例31不同的是，所述上层过滤膜1为亲水过滤膜，所述下层过滤膜2为红细胞凝集素滤膜；具体地：该用于全血过滤的滤膜结构，由两层过滤膜依次从上至下叠加构成；所述过滤膜为多孔结构；所述滤膜结构中叠加的过滤膜的孔径从上至下孔径逐渐变小，面积从上至下逐渐变大；该滤膜结构包括的两层过滤膜分别为上层过滤膜1和下层过滤膜2，所述上层过滤膜1为亲水过滤膜，所述下层过滤膜2为红细胞凝集素滤膜；所述上层过滤膜1的孔径为1μm，所述下层过滤膜2的孔径为0.2～0.5μm；所述上层过滤膜1的厚度为0.5mm，所述下层过滤膜2的厚度为1mm；所述红细胞凝集素滤膜为经过红细胞凝集素处理后的过滤膜，其中均匀分布有红细胞凝集素，所述红细胞凝集素的重量为100ng。
滤膜结构不少于两层过滤膜，也可以叠加更多层过滤膜；过滤膜均为多孔结构，且最上层为疏松多孔结构，即最上层的孔径最大，滤膜结构中叠加的过滤膜从上往下孔径依次逐渐减小，这样的设置便于层层过滤，便于血浆或血清通过，而使血细胞与其他杂质阻留；滤膜结构中叠加的过滤膜从上至下的面积可以是相等的，也可以是从上至下逐渐变大，这样的设置有利于层层过滤，保证上层漏掉的血细胞与其他杂质可以由下层继续过滤。
以上显示和描述了本发明的基本原理、主要特征及优点。本行业的技术人员应该了解，本发明不受上述实施例的限制，上述实施例和说明书中描述的只是说明本发明的原理，在不脱离本发明精神和范围的前提下，本发明还会有各种变化和改进，例如改变过滤膜的材料或过滤膜的孔径等，这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等效物界定。

Claims (15)

  1. 一种全血过滤的方法，其特征在于，具体包括以下步骤：
    (1)选择由至少两层过滤膜依次从上至下叠加构成滤膜结构，且将所述滤膜结构进行红细胞凝集素处理，待用；
    (2)将全血样本加入所述滤膜结构进行过滤；
    (3)收集过滤后的血清或血浆。
  2. 根据权利要求1所述的全血过滤的方法，其特征在于，所述过滤膜为多孔结构；所述滤膜结构中叠加的过滤膜从上至下的孔径逐渐变小，面积逐渐变大或相等。
  3. 根据权利要求1所述的全血过滤的方法，其特征在于，所述滤膜结构包括两层过滤膜，分别为上层过滤膜和下层过滤膜，所述上层过滤膜为红细胞凝集素滤膜，所述下层过滤膜由至少一层亲水性微孔膜叠加组成；或所述上层过滤膜为亲水过滤膜，所述下层过滤膜为红细胞凝集素滤膜；所述红细胞凝集素滤膜中均匀分布有红细胞凝集素。
  4. 根据权利要求3所述的全血过滤的方法，其特征在于，所述上层过滤膜为红细胞凝集素滤膜；所述下层过滤膜为若干滤孔均匀分布且孔径大小不同的过滤膜，或所述下层过滤膜由第一下层膜和第二下层膜依次上下叠加组成，所述第一下层膜的孔径大于所述第二下层膜的孔径。
  5. 根据权利要求3所述的全血过滤的方法，其特征在于，所述上层过滤膜为玻璃纤维滤纸或硝酸纤维素膜或聚砜膜；所述下层过滤膜为亲水性微孔膜，包括玻璃纤维滤纸或硝酸纤维素膜或聚砜膜或乙酸纤维素膜；所述步骤(2)过滤时采用上部加压或下部抽吸加快过滤速度。
  6. 根据权利要求3所述的全血过滤的方法，其特征在于，所述红细胞凝集素滤膜是将过滤膜进行红细胞凝集素处理得到的，其制备过程为：
    1)将红细胞凝集素放入红细胞凝集素缓冲液中稀释；
    2)将所述过滤膜放入步骤1)的含有红细胞凝集素的红细胞凝集素缓冲液中润湿，然后在室温晾干，再放置于37℃～55℃温度烘箱中烘干2h以上；或将在红细胞凝集素缓冲液中润湿后的所述过滤膜进行真空抽干或冻干处理。
  7. 根据权利要求6所述的全血过滤的方法，其特征在于，所述红细胞凝集素缓 冲液为PB或Tris-HCl或CB；所述红细胞凝集素缓冲液的浓度为5mM～1M；所述红细胞凝集素滤膜中添加的红细胞凝集素的重量不少于5ng。
  8. 根据权利要求3所述的全血过滤的方法，其特征在于，所述过滤膜在使用前需采用缓冲液进行处理，所述缓冲液为溶解BSA、氨基酸、含有表面活动性剂成分的任一种缓冲液；处理方法为：将所述过滤膜在缓冲液中润湿后，在室温晾干且干燥2h以上，待用。
  9. 根据权利要求6所述的全血过滤的方法，其特征在于，所述上层过滤膜的孔径不小于0.45μm，所述下层过滤膜的孔径为0.2μm～4μm。
  10. 根据权利要求9所述的全血过滤的方法，其特征在于，所述上层过滤膜的孔径为1μm～5μm；所述上层过滤膜的厚度为0.5～20mm，所述下层过滤膜的厚度为0.05～2mm；红细胞凝集素滤膜中添加的红细胞凝集素的重量为20ng～100ng。
  11. 一种用于全血过滤的滤膜结构，其特征在于，该滤膜结构由至少两层过滤膜依次从上至下叠加构成；所述过滤膜为多孔结构；该滤膜结构中叠加的过滤膜的孔径从上至下孔径逐渐变小，面积从上至下逐渐变大或相等。
  12. 根据权利要求11所述的用于全血过滤的滤膜结构，其特征在于，该滤膜结构包括两层过滤膜，分别为上层过滤膜和下层过滤膜，所述上层过滤膜为红细胞凝集素滤膜，所述下层过滤膜由至少一层亲水性微孔膜叠加组成；或所述上层过滤膜为亲水过滤膜，所述下层过滤膜为红细胞凝集素滤膜；所述红细胞凝集素滤膜中均匀分布有红细胞凝集素。
  13. 根据权利要求12所述的用于全血过滤的滤膜结构，其特征在于，所述上层过滤膜为红细胞凝集素滤膜；所述下层过滤膜为若干滤孔均匀分布且孔径大小不同的过滤膜，或所述下层过滤膜由第一下层膜和第二下层膜依次上下叠加组成，所述第一下层膜的孔径大于所述第二下层膜的孔径。
  14. 根据权利要求12所述的用于全血过滤的滤膜结构，其特征在于，所述上层过滤膜的孔径不小于0.45μm，所述下层过滤膜的孔径为0.2μm～4μm。
  15. 根据权利要求12所述的用于全血过滤的滤膜结构，其特征在于，所述上层过滤膜的孔径为1μm～5μm；所述上层过滤膜的厚度为0.5～20mm，所述下层过滤膜的厚度为0.05～2mm；所述红细胞凝集素滤膜中添加的红细胞凝集素的重量为20ng～100ng。
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CN111308070A (zh) * 2020-02-28 2020-06-19 厦门宝太生物科技有限公司 一种用于从全血中分离红细胞的试剂盒及其从全血中分离红细胞的方法
CN112452367B (zh) * 2020-12-10 2022-02-22 深圳先进技术研究院 一种双层微孔芯片、双层微孔芯片制备方法及生物装置
WO2023200703A1 (en) * 2022-04-15 2023-10-19 Siemens Healthcare Diagnostics Inc. Creatinine lateral flow assay devices and methods of production and use thereof
CN115112463B (zh) * 2022-06-15 2024-08-30 安徽九陆生物科技有限公司 一种微量血液过滤装置及其在血液营养元素检测中的应用
CN115184620B (zh) * 2022-09-14 2023-01-24 山东子峰生物技术有限公司 一种pla2r抗体的量子点荧光检测试纸条、试剂盒及其应用
CN115825434B (zh) * 2022-09-30 2023-08-22 杭州瑞测生物技术有限公司 一种抗原抗体联检的可凝集猫红细胞的猫艾滋检测试剂卡及其检测方法
WO2024086358A1 (en) * 2022-10-20 2024-04-25 Porex Technologies Corporation Separation media and methods of use
CN116625772A (zh) * 2023-05-25 2023-08-22 安徽九陆生物科技有限公司 一种微量血液过滤的方法及应用

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN2240724Y (zh) * 1995-08-09 1996-11-20 中国科学院大连化学物理研究所 用于临床血液成分化验分析的血浆分离器
CN1169886A (zh) * 1996-01-19 1998-01-14 富士胶片公司 血液过滤器
CN1243251A (zh) * 1998-07-28 2000-02-02 华美生物工程公司郑州分公司 全血免疫检测试剂中红细胞和血清的分离方法
US20020036170A1 (en) * 2000-08-11 2002-03-28 Harvey Michael A. Lateral flower plasma separation device
US20140263059A1 (en) * 2013-03-14 2014-09-18 Lawrence J. Burg Plasma separation from blood using a filtration device and methods thereof
CN105307758A (zh) * 2013-06-27 2016-02-03 曼·胡默尔有限公司 陶瓷全血中空纤维膜过滤介质及其用于自全血分离血浆/血清的用途
CN105445083A (zh) * 2015-12-21 2016-03-30 丹娜（天津）生物科技有限公司 免离心全血处理模块
CN106840828A (zh) * 2017-03-29 2017-06-13 天津中新科炬生物制药股份有限公司 一种微量全血中快速分离血浆的方法及分离装置
CN107666961A (zh) * 2015-01-14 2018-02-06 伯乐实验室公司 血液分析系统和方法

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3146163A (en) * 1962-01-23 1964-08-25 John H Brewer Apparatus for separating certain components from blood
DE3729001A1 (de) * 1987-08-31 1989-03-09 Behringwerke Ag Vorrichtung und verfahren zur abtrennung von blutzellen aus koerperfluessigkeiten, die erythrozyten enthalten, und deren verwendung
JP4006551B2 (ja) * 1998-01-29 2007-11-14 東洋紡績株式会社 血漿あるいは血清分離フィルター
JP2004294388A (ja) * 2003-03-10 2004-10-21 Arkray Inc 成分分析装置
JP4298555B2 (ja) * 2004-03-26 2009-07-22 栄研化学株式会社 血液分離法及び装置
FR3012972A1 (fr) * 2013-11-14 2015-05-15 Biomerieux Sa Nouveau milieu filtrant pour l'obtention de plasma, dispositif et procede de filtration associes
MX2017001368A (es) * 2014-08-01 2017-05-11 Siemens Healthcare Diagnostics Inc Separacion de plasma asistido por vacio.
CN209968132U (zh) * 2019-04-27 2020-01-21 南京岚煜生物科技有限公司 一种用于全血过滤的滤膜结构

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN2240724Y (zh) * 1995-08-09 1996-11-20 中国科学院大连化学物理研究所 用于临床血液成分化验分析的血浆分离器
CN1169886A (zh) * 1996-01-19 1998-01-14 富士胶片公司 血液过滤器
CN1243251A (zh) * 1998-07-28 2000-02-02 华美生物工程公司郑州分公司 全血免疫检测试剂中红细胞和血清的分离方法
US20020036170A1 (en) * 2000-08-11 2002-03-28 Harvey Michael A. Lateral flower plasma separation device
US20140263059A1 (en) * 2013-03-14 2014-09-18 Lawrence J. Burg Plasma separation from blood using a filtration device and methods thereof
CN105307758A (zh) * 2013-06-27 2016-02-03 曼·胡默尔有限公司 陶瓷全血中空纤维膜过滤介质及其用于自全血分离血浆/血清的用途
CN107666961A (zh) * 2015-01-14 2018-02-06 伯乐实验室公司 血液分析系统和方法
CN105445083A (zh) * 2015-12-21 2016-03-30 丹娜（天津）生物科技有限公司 免离心全血处理模块
CN106840828A (zh) * 2017-03-29 2017-06-13 天津中新科炬生物制药股份有限公司 一种微量全血中快速分离血浆的方法及分离装置

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP3808434A4

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