CN106840828B - Method and device for quickly separating plasma from trace whole blood - Google Patents
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Abstract
The invention provides a method and a separation device for rapidly separating plasma from trace whole blood, which reduces the physical accumulation of red blood cells in the separation process by overlapping and adsorbing samples from bottom to top, selects a polyether sulfone resin (PES) membrane with an aperture of 3 mu m and an asymmetric structure through sulfonation hydrophilic treatment, effectively isolates the red blood cells and improves the plasma transmittance, and a plasma adsorption area of low-density glass fibers is convenient for absorbing the separated plasma, thereby improving the absorption amount of the plasma; the used separation device comprises a plasma absorption material in an area A, a polyether sulfone resin (PES) membrane in an area B, mixed fibers in an area C and glass fibers in an area D, external equipment is not needed, the separation device is more portable in application, erythrocytes are damaged by inorganic mechanical force, samples are overlapped and adsorbed from bottom to top, the physical accumulation of the erythrocytes in the separation process is reduced, and the plasma absorption area of the low-density glass fibers is convenient for absorbing separated plasma and efficiently separates high-purity plasma in a short time.
Description
Technical Field
The invention relates to the technical field of biological sample separation, in particular to a method and a separation device for quickly separating plasma from trace whole blood.
Background
In the current stage, for the separation of the plasma, the whole blood containing the anticoagulant is kept stand, and sedimentation separation is carried out under the gravity action of red blood cells to obtain supernatant which is the plasma; or centrifuging the whole blood sample containing the anticoagulant, and separating to obtain supernatant as plasma; CN201380041858.6 patent uses a membrane and a capillary tube to separate blood plasma; CN201310254336.6 patent of invention uses PDMS chip to separate plasma through chemically treated microchannel.
For the above prior art means, there are often the following disadvantages:
the standing separation requires a long time for separating the plasma.
Although centrifugal separation reduces separation time, plasma separation can be realized only by means of a centrifuge after sample collection, the centrifuge needs to be installed in a horizontal fixed field and cannot be used in a portable mode, and hemolysis is caused by damage of centrifugal force to red blood cells.
The CN201380041858.6 patent of the invention provides a portable plasma separation device that allows the membrane to be in direct contact with the whole blood sample for vertical filtration. During the filtration process, the red blood cells can settle to the bottom of the membrane due to the action of gravity, so that the small-aperture membrane is blocked, and the separation efficiency is directly influenced.
The microchannel used in the invention of CN201310254336.6 needs complicated chemical pretreatment before it can be used, because the red blood cell system is divided into primitive red blood cell, early red blood cell, middle red blood cell, late red blood cell, reticulocyte and red blood cell, the cell diameter is about 6-10 microns, but the allowable diameter of the microchannel of the device is 0.5-50 microns, which can cause part of red blood cells mixed in the separated plasma during the separation process.
Therefore, the method and the device which can be independently used without a centrifuge or an external instrument and can efficiently separate the high-purity plasma in a short time are found, and the method and the device have important application value.
Disclosure of Invention
The invention aims to provide a method for quickly separating plasma from trace whole blood.
The invention aims to provide a separation device for realizing rapid separation of plasma in trace whole blood.
In order to solve the technical problems, the technical scheme of the invention is as follows:
a method for rapidly separating plasma from trace whole blood comprises the following specific steps:
(1) taking a plasma absorption material, a sulfonated hydrophilic treated polyethersulfone resin (PES) membrane with an asymmetric structure and a pore diameter of 0.03-10 mu m, glass fibers containing 0.1-10 mg/mL erythrocyte monoclonal antibody and 0-2.2mg/mL EDTA-K2 (dipotassium ethylenediamine tetraacetic acid) or EDTA-NA (disodium ethylenediamine tetraacetic acid), glass fibers containing 0.1-10 mg/mL erythrocyte monoclonal antibody and 0-2.2mg/mL EDTA-K2 (dipotassium ethylenediamine tetraacetic acid) or EDTA-NA (disodium ethylenediamine tetraacetic acid) and placing the materials in an edge overlapping way in turn, wherein the plasma absorption material is placed at the uppermost layer and is glass fibers, polyester, mixed fibers or pure cotton,
a plasma absorbing material, designated zone a;
sulfonated hydrophilic treated polyether sulfone resin with asymmetric structure and pore diameter of 0.03-10 microns
(PES) membrane, referred to as zone B;
mixed fiber containing 0.1mg/mL-10mg/mL erythrocyte monoclonal antibody and 0-2.2mg/mL EDTA-K2 (ethylene diamine tetraacetic acid dipotassium) or EDTA-NA (ethylene diamine tetraacetic acid disodium), called zone C;
glass fiber containing 0.1mg/mL-10mg/mL erythrocyte monoclonal antibody and 0-2.2mg/mL EDTA-K2 (dipotassium ethylenediaminetetraacetate) or EDTA-NA (disodium ethylenediaminetetraacetate), called D-domain;
(2) the whole blood sample firstly reacts with the glass fiber of the D area precoated erythrocyte monoclonal antibody with the concentration of 0.1mg/mL-10mg/mL and the EDTA anticoagulant of 0-2.2mg/mL, the EDTA ensures that the whole blood can not agglutinate in the separation process, the erythrocytes in the whole blood can generate specific immunological reaction with the erythrocyte monoclonal antibody, and most of the erythrocytes can be adsorbed in the D area due to the irregular structure of the glass fiber;
(3) a small part of red blood cells and plasma react with mixed fibers of a red blood cell monoclonal antibody and an EDTA anticoagulant, which are pre-coated in the C area at the concentration of 0.1-10 mg/mL, and most of red blood cells are adsorbed through the reaction of the D area and the C area;
(4) a small amount of free red blood cells are trapped in the zone C by the polyethersulfone resin membrane in the zone B through the physical action of the molecular sieve, and only plasma can pass through and is finally adsorbed in the plasma absorbing material in the zone A.
Preferably, in the method for rapidly separating plasma from a trace amount of whole blood, the pore diameter of the polyethersulfone resin membrane is 3 μm.
Preferably, in the method for rapidly separating plasma from trace whole blood, the glass fiber in the C area contains 2.2mg/mL of EDTA-K2.
Preferably, in the method for rapidly separating plasma from a trace amount of whole blood, the glass fiber in the D region contains 0.1mg/mL of erythrocyte monoclonal antibody.
A separation device for realizing rapid separation of plasma in trace whole blood comprises an A-area plasma absorption material, a B-area polyether sulfone resin (PES) membrane, a C-area mixed fiber, a D-area glass fiber, a PVC plate and a shell, wherein the PVC plate and the shell are matched and fixed to form a box-shaped hollow structure, the A-area plasma absorption material, the B-area polyether sulfone resin (PES) membrane, the C-area mixed fiber and the D-area glass fiber are arranged in the box-shaped hollow structure and are sequentially overlapped on the PVC plate in an edge overlapping mode, the A-area plasma absorption material is arranged on the uppermost layer, one side, which is not overlapped with the B-area polyether sulfone resin membrane, of the A-area plasma absorption material is called a plasma temporary storage pool, one side, which is close to the overlapping with the B-area polyether sulfone resin membrane, of the A-area plasma absorption material is called a plasma absorption area, three through holes are formed in the shell and are correspondingly arranged right, wherein the content of the first and second substances,
the plasma absorption material in the area A is glass fiber, polyester, mixed fiber or pure cotton filter paper,
the polyethersulfone resin (PES) membrane in the B area is a sulfonated and hydrophilically treated polyethersulfone resin membrane with an asymmetric structure and a pore diameter of 0.03-10 mu m,
the glass fiber in the C area is a mixed fiber containing 0.1mg/mL-10mg/mL erythrocyte monoclonal antibody and 0-2.2mg/mL EDTA-K2 (ethylene diamine tetraacetic acid dipotassium) or EDTA-NA (ethylene diamine tetraacetic acid disodium),
the D-region glass fiber is glass fiber containing 0.1mg/mL-10mg/mL erythrocyte monoclonal antibody and 0-2.2mg/mL EDTA-K2 (ethylene diamine tetraacetic acid dipotassium) or EDTA-NA (ethylene diamine tetraacetic acid disodium).
Preferably, in the separation device for rapidly separating plasma from trace whole blood, the pore diameter of the polyethersulfone resin membrane is 3 μm.
Preferably, the separation device for realizing rapid separation of plasma in trace whole blood comprises 2.2mg/mL EDTA-K2 in the glass fiber in the C area.
Preferably, the separation device for realizing rapid separation of plasma from trace whole blood comprises the D-zone glass fiber containing 0.1mg/mL erythrocyte monoclonal antibody.
The invention has the beneficial effects that:
according to the method for rapidly separating the plasma from the trace whole blood, physical accumulation of red blood cells in the separation process is reduced through overlapping adsorption of samples from bottom to top, a Polyethersulfone (PES) membrane with an aperture of 3 mu m and a sulfonated hydrophilic treatment asymmetric structure is selected, the red blood cells are effectively isolated, the plasma transmittance is improved, a plasma adsorption area of low-density glass fibers is convenient for absorbing the separated plasma, and the absorption amount of the plasma is improved; the separation device has the advantages of small volume, low production cost, no need of external equipment, more portability in application, no mechanical force to destroy red blood cells, reduction in physical accumulation of red blood cells in the separation process due to overlapped adsorption of samples from bottom to top, convenience for absorbing separated plasma by the plasma adsorption area of the low-density glass fiber, and high-purity plasma separation in a short time.
Drawings
FIG. 1 is a schematic diagram of the internal structure of the separation device for realizing rapid separation of plasma in trace whole blood according to the invention; in the figure, 1-S1 pad 2-S2 pad 3-PES membrane 4-AB pad
Fig. 2 is a schematic diagram of the external structure of the separation device for realizing rapid separation of plasma in trace whole blood, and A, B, C three through holes are arranged on the plastic card.
Detailed Description
The technical solution of the present invention is further described with reference to the following specific examples.
The "erythrocyte monoclonal antibody" mentioned in the following examples refers to a murine anti-human erythrocyte monoclonal antibody, which is a commercial biological material purchased from Shenzhen Shenpeng biological corporation.
Example 1
1. Materials:
erythrocyte monoclonal antibody, EDTA-K2, mixed fiber, glass fiber, PES films of 0.03 μm, 3.00 μm and 10.00 μm, PVC plate, EDTA-K2 anticoagulated whole blood sample P4 (the whole blood sample P4 is a negative whole blood sample, a negative whole blood sample from Tianjin Huatai Hospital) of known hematocrit.
2. Method of producing a composite material
Soaking the mixed fiber and glass fiber in 0.01M PH7.4PBS buffer solution containing 0.1mg/mL erythrocyte monoclonal antibody and 2.2mg/mL LEDTA-K2, fully soaking, and oven drying at 45 deg.C for 12 hr. The treated glass fibers were designated as S1 mat, the treated hybrid fibers were designated as S2 mat, and the untreated glass fibers were designated as AB mat.
As shown in fig. 1 and 2, 4 materials are sequentially overlapped and adhered on a PVC plate according to the sequence of an S1 pad 1, an S2 pad 2, a PES membrane 3 and an AB pad 4, and 3 semi-finished products are formed according to the difference of the PES membrane. And cutting the semi-finished product into strips of 4mm, and packaging the strips into plastic cards to form the separating device. Mould and be equipped with three through-hole A hole, B hole and C hole on the card, wherein, the C hole sets up directly over the S1 pad, and A hole and B hole set up directly over the AB pad, and A hole and C hole set up respectively in the both sides in B hole simultaneously.
The P4 sample was chosen to test the separation efficiency of the separation device. The separation rate of plasma was calculated according to the formula separation efficiency (1-packed erythrocyte) x sample amount divided by separation volume.
3. Results
The experimental results are shown in Table 1, and show that the separation efficiency is affected by the small pore size of 0.03 μm PES membrane, the separation efficiency of plasma is highest when the pore size of 10 μm PES membrane is large, but a part of erythrocytes seep into the separated plasma due to the large pore size, and the infiltration of free erythrocytes can be sufficiently isolated by the 3 μm PES membrane, and the separation efficiency is also high.
TABLE 1
Example 2
1. Materials:
erythrocyte monoclonal antibody, EDTA-K2, mixed fiber, glass fiber, 3 μm PES membrane, PVC plate, EDTA-K2 anticoagulant whole blood sample P4 of known hematocrit.
2. Method of producing a composite material
Soaking the mixed fiber and glass fiber in 0.01M PH7.4PBS buffer solution containing 0.1mg/mL erythrocyte monoclonal antibody and 2.2mg/mL LEDTA-K2, fully soaking, and oven drying at 45 deg.C for 12 hr. The treated glass fibers were designated as S1 mat, the treated hybrid fibers were designated as S2 mat, and the untreated glass fibers were designated as AB mat.
And (3) sequentially overlapping and sticking 4 materials on the PVC plate according to the sequence of the S1 pad, the S2 pad, the PES film and the AB pad to form a semi-finished product. The semi-finished product was cut into strips of 2mm, 4mm and 8mm, respectively, and loaded into plastic cards of different sizes to form separation devices (see example 1 for structure). The P4 sample is selected to test the separation efficiency of the separation device. After 50, 100 and 200 μ L of whole blood samples were added to the C well of the separation device, respectively, and left to stand for 3 minutes, plasma was aspirated from the B well of the separation device using a pipette and the volume of plasma was measured, and the separation efficiency was calculated according to the formula (1-packed erythrocyte volume) × sample volume ÷ separation volume).
3. Results
The results are shown in tables 2-4 and show that the separation efficiency was 60.57% at the highest when 50. mu.L of the sample was separated by a 2mm separation device, and the excess sample remained only in the well. The separation efficiency of the 4mm separation device for separating 100. mu.L of sample is 61.15% at the maximum, the separation device cannot separate the sample due to the small amount of the adsorptive material sample of the separation device, and the excessive sample only stays in the sample adding hole. The separation efficiency of the 8mm separation device for separating 200 mu L of samples is 63.48% at most, the separation device cannot separate the samples due to the small sample amount of the adsorptive material of the separation device, and the sample amount is insufficient, so that the separation efficiency of the samples is affected.
TABLE 22 mm detection results of the separation device
TABLE 34 mm detection results of the separation device
TABLE 48 mm detection result of the separation device
Example 3
1. Materials:
erythrocyte monoclonal antibody, EDTA-K2, low-density glass fiber, high-density glass fiber, 3 μm PES membrane, PVC plate, negative EDTA-K2 anticoagulated whole blood samples P1, P2, P3, P4 and P5 (all negative whole blood samples from Tianjin Huatai hospital) with known hematocrit.
2. Method of producing a composite material
Soaking the mixed fiber and glass fiber in 0.01M PH7.4PBS buffer solution containing 0.1mg/mL erythrocyte monoclonal antibody and 2.2mg/mL LEDTA-K2, fully soaking, and oven drying at 45 deg.C for 12 hr. The treated glass fibers were designated as S1 mat, the treated hybrid fibers were designated as S2 mat, and the untreated glass fibers were designated as AB mat.
And (3) sequentially overlapping and sticking 4 materials on the PVC plate according to the sequence of the S1 pad, the S2 pad, the PES film and the AB pad to form a semi-finished product. The semi-finished product was cut into 4mm strips and loaded into plastic cards to form separation devices (see example 1 for structure). And selecting EDTA-K2 anticoagulated whole blood sample with known hematocrit to detect the separation efficiency of the separation device. The separation rate of plasma was calculated according to the formula separation efficiency (1-packed erythrocyte) x sample amount divided by separation volume.
3. Results
The results are shown in Table 5, and show that the blood sample with hematocrit in the range of 0.38-0.51 has plasma separating efficiency of about 62.46% after being separated by the plasma separating device, and the plasma separating device consisting of 3 μm PES membrane, S1 pad containing 0.1mg/mL erythrocyte monoclonal antibody, S2 pad containing 2.2mg/mL EDTA-K2 and AB pad is suitable for blood samples with different hematocrit.
TABLE 5
The above detailed description of the method for rapidly separating plasma from a trace amount of whole blood and the separation device with reference to the embodiments is illustrative and not restrictive, and several embodiments can be enumerated according to the limited scope, so that changes and modifications without departing from the general concept of the present invention shall fall within the protection scope of the present invention.
Claims (6)
1. A method for rapidly separating plasma from trace whole blood is characterized in that: the method comprises the following specific steps: (1) collecting plasma absorbing material, sulfonated hydrophilic polyethersulfone resin membrane with asymmetric structure and pore diameter of 0.03-10 μm, erythrocyte monoclonal antibody of 0.1-10 mg/mL and 2.2mg/mLEDTA-K2Or EDTA-Na2The mixed fiber contains 0.1mg/mL-10mg/mL erythrocyte monoclonal antibody and 2.2mg/mLEDTA-K2Or EDTA-Na2The glass fibers are overlapped at the edges in sequence, the plasma absorption material is arranged at the uppermost layer, the plasma absorption material is glass fiber, polyester, mixed fiber or pure cotton filter paper,
wherein, the plasma absorption material is called as A area;
the sulfonated and hydrophilically treated polyethersulfone resin film with an asymmetric structure and a pore diameter of 0.03-10 mu m is called as a B area;
contains 0.1mg/mL-10mg/mL erythrocyte monoclonal antibody and 2.2mg/mL EDTA-K2 or EDTA-Na2The mixed fiber of (3), referred to as zone C;
contains 0.1mg/mL-10mg/mL erythrocyte monoclonal antibody and 2.2mg/mLEDTA-K2Or EDTA-Na2The glass fiber of (a), referred to as zone D;
(2) whole blood sample first and zone D precoated 0.1Erythrocyte monoclonal antibody with concentration of mg/mL-10mg/mL and 2.2mg/mLEDTA-K2Or EDTA-Na2Glass fibre reaction of (D), EDTA-K2Or EDTA-Na2The agglutination of the whole blood is avoided in the separation process, the erythrocyte in the whole blood and the erythrocyte monoclonal antibody are subjected to specific immunological reaction, and most of the erythrocyte is adsorbed in the D zone due to the irregular structure of the glass fiber;
(3) less part of red blood cells and plasma are pre-coated with red blood cell monoclonal antibody and EDTA-K with concentration of 0.1mg/mL-10mg/mL and C area2Or EDTA-Na2The mixed fiber in the D area reacts with the mixed fiber in the C area to adsorb most of red blood cells, and the mixed fiber material with flowability and water absorption stronger than those of the glass fiber in the D area is selected to promote the rapid separation of the red blood cells and the blood plasma;
(4) a small amount of free red blood cells are trapped in the zone C by the polyethersulfone resin membrane in the zone B through the physical action of the molecular sieve, and only plasma can pass through and is finally adsorbed in the plasma absorbing material in the zone A.
2. The method for rapidly separating plasma from trace whole blood according to claim 1, wherein: the pore diameter of the polyether sulfone resin film is 3 mu m.
3. The method for rapidly separating plasma from trace whole blood according to claim 1, wherein: the glass fiber of the D area contains 0.1mg/mL erythrocyte monoclonal antibody.
4. A separation device for achieving rapid separation of plasma from trace whole blood according to any one of claims 1 to 3, wherein: the plasma absorption material of the area A, the polyether sulfone resin film of the area B, the mixed fiber of the area C, the glass fiber of the area D, a PVC plate and a shell are matched and fixed to form a box-shaped hollow structure, the plasma absorption material of the area A, the polyether sulfone resin film of the area B, the mixed fiber of the area C and the glass fiber of the area D are arranged in the box-shaped hollow structure and are sequentially overlapped on the edges of the box-shaped hollow structure, the plasma absorption material of the area A is arranged on the uppermost layer, the side, which is not overlapped with the polyether sulfone resin film of the area B, of the plasma absorption material of the area A, the side, which is close to the side, which is overlapped with the polyether sulfone resin film of the area B, of the plasma absorption material of the area A is called a plasma absorption area, three through holes are arranged on the
Wherein the plasma absorption material in the area A is glass fiber, polyester, mixed fiber or pure cotton filter paper,
the polyethersulfone resin film in the B area is a sulfonated and hydrophilically treated polyethersulfone resin film with an asymmetric structure and a pore diameter of 0.03-10 mu m,
the mixed fiber in the C region contains 0.1mg/mL-10mg/mL erythrocyte monoclonal antibody and 2.2mg/mLEDTA-K2Or EDTA-Na2The mixed fiber of (a) and (b),
the D-region glass fiber contains 0.1mg/mL-10mg/mL erythrocyte monoclonal antibody and 2.2mg/mLEDTA-K2Or EDTA-Na2The glass fiber of (1).
5. A separation device for achieving rapid separation of plasma in minute amount of whole blood according to claim 4, wherein: the pore diameter of the polyether sulfone resin film is 3 mu m.
6. A separation device for achieving rapid separation of plasma in minute amount of whole blood according to claim 4, wherein: the D-area glass fiber contains 0.1mg/mL erythrocyte monoclonal antibody.
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| CN108152177B (en) * | 2017-12-11 | 2020-11-24 | 三诺生物传感股份有限公司 | Method for rapidly detecting hematocrit |
| CN109925884A (en) * | 2019-04-27 | 2019-06-25 | 南京岚煜生物科技有限公司 | A kind of method of Whole Blood Filtration and filter membrane structure for Whole Blood Filtration |
| CN116731962B (en) * | 2023-08-14 | 2023-11-03 | 天津中新科炬生物制药股份有限公司 | Kit and method for separating red blood cells from whole blood |
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