Condensate removing device and method

Abstract

The invention relates to the technical field of blood routine examination, and discloses a condensation element removing device and method, and the condensation element removing device comprises a first container, a second container, a filter screen and a carrier. The blood collection tube, the first container and the second container are assembled and uniformly mixed up and down, a detection sample moves back and forth in the blood collection tube and the second container, and when the detection sample flows to the second container from the blood collection tube and the first container, blood cells are intercepted by the filter screen, and blood plasma without the blood cells enters the second container, so that the blood collection tube and the second container are separated from each other. And combining condensed elements in the filtered plasma with an anti-human IgM antibody on the surface of the carrier, fixing the condensed elements on the surface of the carrier, pouring the reacted plasma into a first container and a blood collection tube, fully and uniformly mixing the reacted plasma with blood cells, and carrying out clinical blood routine examination. Theoretically, the process does not cause cell loss, red blood cells do not agglutinate again due to temperature change, and the method is more favorable for helping clinical laboratories to solve the problems of tedious treatment, long time consumption and the like of condensation set samples.

Description

Technical Field

[0001] The present invention relates to the technical field of routine blood testing, and in particular to a device and method for removing cold agglutinins. Background Art

[0002] Cold Agglutinins (CA) are produced by B cells and are IgM autoantibodies, cold-reactive anti-erythrocyte antibodies. CA is commonly seen in people with mycoplasma infection, infectious mononucleosis, multiple myeloma, nonspecific inflammation, autoimmune diseases, cirrhosis, lymphoma, etc. When the temperature is below 31°C, CA easily binds to class I antigens on the surface of red blood cells, causing reversible agglutination of red blood cells. This agglutination phenomenon can easily interfere with blood cell analysis and detection, such as the disproportion between red blood cells (RBC) and hemoglobin (HB), significantly high MCV, block-like agglutination of RBCs in blood smears, and fine sand-like agglutinates in some specimens. In severe cases, the specimens are clotted, making it impossible to test the specimens, affecting clinical condition assessment and treatment. Timely and effective elimination of interference with test results caused by cold agglutination is conducive to providing a more effective basis for clinical treatment.

[0003] It is not uncommon to encounter cold agglutination specimens in clinical routine blood testing, which is more common when the temperature drops. At present, there are two main methods for clinical laboratories to deal with cold agglutination specimens: ① 37℃ water bath, by raising the temperature of the surrounding environment of the specimen to make the agglutination reversible, so that the red blood cells disaggregate, this method usually takes a long time. 37℃ water bath has high requirements for the environment and requires a water bath or incubator. If the testing task is performed in an area with poor conditions, it will affect the test results and testing time. Blood routine is not only a routine test item, but also plays an important role in guiding clinical treatment in emergency rescue, so the impact of environmental factors on the results should be reduced as much as possible. In addition, for samples with severe cold agglutination, once they are out of the 37℃ incubation environment, the agglutination state can be restored immediately, and they still cannot be tested. Therefore, this method is not the best way to solve cold agglutination samples. ②Plasma replacement with saline. The whole blood sample anticoagulated with EDTA-K2 vacuum anticoagulation tube is centrifuged at low speed (500r / min) for 5min, and the upper plasma is replaced with an equal amount of saline. The purpose of removing CA is achieved through plasma replacement. The first is the matrix effect. Different matrices may affect the osmotic pressure of blood cells, causing cell deformation, shrinkage or hemolysis, or other effects caused by different matrices. The test results are the state of blood cells after replacement with saline. In addition, there is also the problem of cell loss during the replacement process, especially platelets. Since the speed of 500r / min cannot make the platelets completely deposit to the bottom, there are still many platelets in the plasma after centrifugation. Therefore, this method is easy to cause platelet loss while removing CA, resulting in a large difference between the platelet results and the actual results. If the speed is increased, it will cause red blood cell destruction and hemolysis. In addition, for specimens with severe agglutination, multiple centrifugation is required for plasma replacement, and the operation process is easy to cause hemolysis. Summary of the invention

[0004] The purpose of the present invention is to provide a device and method for removing cold agglutinins. The present invention proposes a device and method for removing cold agglutinins to solve the problem that cold agglutination leads to abnormal parameters of routine blood test items, and it is impossible to test severe cold agglutination samples, which affects clinical condition assessment and treatment. This method solves the problem that plasma exchange causes platelet loss and excessive centrifugal speed easily leads to blood cell damage. This method also solves the problem that the time required for a 37°C water bath is long, and when the specimen leaves the 37°C water bath environment, it is easy to re-agglutinate, and this method is not ideal for specimens with severe cold agglutination. This method is conducive to the routine blood test of specimens with cold agglutination in clinical practice, and can provide timely and accurate test results for the clinic.

[0005] The above technical purpose of the present invention is achieved through the following technical solutions: a cold agglutinin removal device, comprising a first container, a second container, a filter, and a carrier, the first container is provided with a connecting port, the interior of the first container is connected with the interior of the second container, the filter is located in the first container, the filter is used to intercept blood cells, the second container is used to place the carrier, and the carrier is provided with anti-human IgM antibodies.

[0006] The present invention is further configured as follows: the mesh diameter of the filter is smaller than the diameter of the blood cells.

[0007] By adopting the above technical solution, the function of the filter is to intercept blood cells and filter out plasma that does not contain blood cells. Therefore, in theory, the diameter of the filter mesh is smaller than the diameter of blood cells. Blood cells are mainly divided into red blood cells (RBC), white blood cells (WBC), and platelets (PLT). The diameter of RBC is 7-8.5μm, and WBC is divided into neutrophils (10-12μm), eosinophils (10-15μm), basophils (10-12μm), monocytes (14-20μm) and lymphocytes (6-8μm). The diameter of platelets is 2-4μm. According to the size of the cells, in theory, a filter with a mesh diameter less than 2μm can meet the requirements.

[0008] The present invention is further configured as follows: the mesh diameter of the filter is 1 μm.

[0009] The present invention is further configured as follows: the carrier is a magnetic bead, the diameter of the magnetic bead is larger than the diameter of the filter mesh, the anti-human IgM antibody is coupled to the magnetic bead, and the second container is detachably connected to the first container.

[0010] By adopting the above technical solution, when the diameter of the filter mesh is 1 μm, magnetic beads with a diameter of 3 μm are selected. The magnetic beads cannot reversely penetrate the filter, and the filter is used to isolate blood cells and magnetic beads.

[0011] The present invention is further configured as follows: the method of coupling the magnetic beads to the anti-human IgM antibody is as follows:

[0012] A. Prepare coupling solution: 100 mM MES pH 4.8, blocking solution: 3 M Ethanolamine, 1% BSA, 0.5% T-20, pH 8.0, and storage solution: 0.05% proclin-300 in PBS buffer;

[0013] B. Mix the magnetic beads with a vortex oscillator, place the magnetic beads in an EP tube, add coupling solution to make the final concentration of the magnetic beads 5 mg / mL, mix with a vortex oscillator, and then place on a magnetic separation rack to enrich the magnetic beads, remove the supernatant, and then add coupling solution to repeat the washing once;

[0014] C. Add coupling solution, add activator EDC and NHS to a final concentration of 1.5 mg / mL, add magnetic beads to a final concentration of 5 mg / mL. Shake and mix, then place at 37°C for 30 minutes;

[0015] D. After the reaction is completed, place the magnetic rack, remove the supernatant, and use the coupling solution to prepare the antibody so that the ratio of antibody to magnetic beads is 1:50 and the final concentration of magnetic beads is 5 mg / mL. Shake and mix well, and then place at 37°C for 2 hours to rotate and mix well;

[0016] E. After the reaction is completed, place the beads on a magnetic stand, remove the supernatant, add blocking solution to a concentration of 5 mg / mL, shake and mix, and then place at 37°C for 1 hour.

[0017] F. After blocking is completed, repeat step B and finally store the antibody-coupled magnetic beads for future use.

[0018] The present invention is further configured as follows: the carrier is a honeycomb structure or a porous structure, and the carrier is coated with the anti-human IgM antibody.

[0019] Through the above technical solution, the honeycomb structure or porous structure can provide more contact area to combine more cold agglutinins, thereby achieving a better cold agglutinin removal effect.

[0020] The present invention is further configured as follows: the carrier material is polystyrene, and the method of coating the carrier with the anti-human IgM antibody is as follows:

[0021] A. Coating: Coating with anti-human IgM antibody (μ-chain specific), coating solution: pH 9.6, 0.01M carbonate buffer, incubate at 37°C for 2 hours.

[0022] B. Washing: Wash with PBS buffer for 3 times and spin dry.

[0023] C. Blocking: Add blocking solution and incubate at 37°C for 2 hours.

[0024] By adopting the above technical solution, the advantage of polystyrene material is its strong plasticity, which makes it convenient to make a carrier with a porous structure.

[0025] The present invention is further configured as follows: the first container is made of polyethylene terephthalate and / or the connection port is embedded with an inner ring of latex material.

[0026] By adopting the above technical solution, the size of the inner ring matches the tube opening of a commonly used blood collection tube and is used for docking with the blood collection tube.

[0027] The present invention is further configured as follows: the first container shape and / or the second container shape is cylindrical.

[0028] The present invention also provides a method for removing cold agglutinins, comprising the following steps:

[0029] Step 1: couple the anti-human IgM antibody to the magnetic bead carrier, and according to the severity of the cold agglutination of the test sample, take an appropriate amount of magnetic beads and place them in the second container; or coat the polystyrene carrier in the second container with the anti-human IgM antibody;

[0030] Step 2, assembling the second container containing the carrier with the first container, and assembling the blood collection tube containing the test sample with the connecting port of the first container;

[0031] Step 3, shaking the assembled blood collection tube, the first container, and the second container up and down to make the plasma go back and forth between the blood collection tube and the second container to allow it to fully react until there is no obvious agglutination phenomenon;

[0032] Step 4, placing the blood collection tube at the bottom and the second container at the top, centrifuging instantly to return the plasma to the blood collection tube;

[0033] Step 5. Remove the blood collection tube, cover the tube cap, shake well, and perform a routine blood test.

[0034] By adopting the above technical solution,

[0035] The beneficial effects of the present invention are:

[0036] 1. For the detection sample of cold agglutination, the present invention assembles the blood collection tube with the first container and the second container and mixes them up and down. The detection sample goes back and forth between the blood collection tube and the second container. When the detection sample flows from the blood collection tube and the first container to the second container, the blood cells are intercepted by the filter, and the plasma without blood cells enters the second container. The filtered plasma reacts with the carrier coated with anti-human IgM antibodies, and the cold agglutinin in the plasma binds to the anti-human IgM antibodies on the surface of the carrier, thereby being fixed on the surface of the carrier, and then the reacted plasma is poured into the first container and the blood collection tube, and clinical blood routine tests are performed after being fully mixed with blood cells. In theory, this process will not cause cell loss, and red blood cells will not re-agglutinate due to temperature changes. It is more conducive to helping clinical laboratories solve the problems of cumbersome and time-consuming cold agglutination sample processing.

[0037] 2. The present invention is based on the two aspects of not relying on the environment and not performing plasma exchange to cause cell loss. The method based on antigen-antibody immunity combined with the corresponding carrier realizes the removal of cold agglutinins, which fundamentally solves the problem. It avoids the problem of incubation or water bath at 37°C and agglutination after the environment changes. It also avoids repeated centrifugation of plasma exchange, insufficient or excessive centrifugal force causing cell damage and loss, and avoids the influence of plasma exchange and matrix effect on the cell environment.

[0038] 3. The device of the present invention describes two methods for separating cold agglutinins, one is to use polystyrene coated anti-human IgM antibodies, and the other is to couple anti-human IgM antibodies through magnetic beads. The above two methods combined with the device of the present invention can achieve the optimization of the processing of cold agglutinin samples. The reaction tube made of polystyrene coated anti-human IgM antibodies has the following advantages for processing cold agglutinin samples: ① After polystyrene is coated with anti-human IgM antibodies, cold agglutinins composed of IgM can be adsorbed; ② During the reaction process, the release of blood cell agglutination can be visually observed, and the specimen can be tested in time when the agglutination is released; ③ It will not cause cell damage and loss; ④ The size of this device is similar to that of a blood collection tube, which is convenient to carry, operate and store. The use of magnetic bead-coupled anti-human IgM antibodies to treat cold agglutination samples has the following advantages: ① The magnetic bead-coupled anti-human IgM antibodies are configured in advance for use, and the degree of agglutination is observed with the naked eye. According to the degree of agglutination, an appropriate amount of magnetic beads are added to the device for reaction, which is convenient for controlling the dosage; ② During the reaction, the release of blood cell agglutination can be visually observed, and the specimen can be tested in time when the agglutination is released; ③ It will not cause damage or loss to the cells; ④ After configuration, the magnetic bead-coupled anti-human IgM antibodies are placed separately, which is easy to store, carry and use. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 It is a structural schematic diagram of Example 1.

[0040] Figure 2 It is a schematic diagram of the separation state between the first container and the second container in Example 1.

[0041] Figure 3 It is a schematic diagram of the positional relationship between the first container and the filter screen in Example 1.

[0042] Figure 4 It is a schematic diagram of the positional relationship between the second container and the carrier in Example 1.

[0043] Figure 5 It is a schematic diagram of the separation state between the first container and the second container of Example 2.

[0044] In the figure, 1, first container; 11, connecting port; 12, inner ring; 2, second container; 3, filter screen; 4, carrier. DETAILED DESCRIPTION

[0045] Embodiment 1: A cold agglutinin removal device, such as Figures 1 to 4As shown, it includes a first container 1, a second container 2, a filter 3, and a carrier 4. The first container 1 and the second container 2 are both cylindrical in shape. The first container 1 is made of PET plastic. One end of the first container 1 is a connection port 11, and an inner ring 12 made of latex is embedded at the connection port 11. The size of the inner ring 12 matches the tube opening of the blood collection tube commonly used in the market, and is used to connect to the tube opening of the blood collection tube. The tube opening at the other end of the first container 1 is also an open structure, and is also embedded with an inner ring made of latex, which is used to connect to the second container 2.

[0046] The second container 2 is made of polystyrene, one end of which is open and inserted into the tube of the first container 1; the other end of which is a closed structure. The carrier is made of polystyrene and has a honeycomb structure, which is fixed in the second container 2. The carrier 4 is coated with anti-human IgM antibodies, and the method of coating the carrier with the anti-human IgM antibodies is as follows:

[0047] A. Coating: Coating with anti-human IgM antibody (μ-chain specific), coating solution: pH 9.6, 0.01M carbonate buffer, incubate at 37°C for 2 hours.

[0048] B. Washing: Wash with PBS buffer for 3 times and spin dry.

[0049] C. Blocking: Add blocking solution and incubate at 37°C for 2 hours.

[0050] The filter 3 is fixed in the middle of the first container 1 and has a mesh diameter of 1 μm, and is used to intercept blood cells.

[0051] A method for removing cold agglutinins using the cold agglutinin removal device comprises the following steps:

[0052] Step 1: coating the polystyrene honeycomb structure carrier 4 in the second container 2 with anti-human IgM antibodies;

[0053] Step 2: Assemble the second container 2 containing the carrier 4 with the first container 1, remove the cap of the blood collection tube containing the test sample, connect it to the inner ring 12 of the connecting port of the first container 1, and assemble them together so that the blood collection tube, the first container 1, and the second container 2 form a closed tube cavity;

[0054] Step three, shake the assembled blood collection tube, first container 1, and second container 2 up and down. Shaking up and down means gently turning them upside down. You can use a laboratory shaking device or turn them upside down manually. During the inversion process, the plasma enters the second container 2 through the filter, and the cold agglutinin in the plasma combines with the anti-human IgM antibody coated with the carrier 4 in the second container 2. The plasma with the cold agglutinin removed passes through the filter and returns to the blood collection tube. During this process, observe the blood in the blood collection tube, and when the agglutination phenomenon is resolved, it is judged that there is no obvious agglutination phenomenon;

[0055] Step 4: Place the blood collection tube at the bottom and the second container 2 at the top, so that all the plasma returns to the blood collection tube, and centrifuge instantly to return the plasma to the blood collection tube;

[0056] Step 5. Remove the blood collection tube, cover the tube cap, shake well, and perform a routine blood test.

[0057] Embodiment 2: A cold agglutinin removal device, such as Figure 5 As shown, it includes a first container 1, a second container 2, a filter 3, and a carrier 4. The first container 1 and the second container 2 are both cylindrical in shape. The first container 1 is made of PET plastic. One end of the first container 1 is a connection port 11, and an inner ring 12 made of latex is embedded at the connection port 11. The size of the inner ring 12 matches the tube opening of the blood collection tube commonly used in the market, and is used to connect with the tube opening of the blood collection tube. The tube opening at the other end of the first container 1 is also an open structure, and is also embedded with an inner ring 12 made of latex, which is used to connect with the second container 2.

[0058] The second container 2 is made of PET plastic, one end of which is open for insertion into the tube of the first container 1, and the other end of which is a closed structure. The carrier 4 is a magnetic bead, and the selected magnetic bead is 3 μm.

[0059] The method for coupling the anti-human IgM antibody with magnetic beads is as follows:

[0060] A. Prepare coupling solution: 100 mM MES pH 4.8, blocking solution: 3 M Ethanolamine, 1% BSA, 0.5% T-20, pH 8.0, and storage solution: 0.05% proclin-300 in PBS buffer;

[0061] B. Mix the magnetic beads with a vortex oscillator, place the magnetic beads in an EP tube, add coupling solution to make the final concentration of the magnetic beads 5 mg / mL, mix with a vortex oscillator, and then place on a magnetic separation rack to enrich the magnetic beads, remove the supernatant, and then add coupling solution to repeat the washing once;

[0062] C. Add coupling solution, add activator EDC and NHS to a final concentration of 1.5 mg / mL, add magnetic beads to a final concentration of 5 mg / mL. Shake and mix, then place at 37°C for 30 minutes;

[0063] D. After the reaction is completed, place the magnetic rack, remove the supernatant, and use the coupling solution to prepare the antibody so that the ratio of antibody to magnetic beads is 1:50 and the final concentration of magnetic beads is 5 mg / mL. Shake and mix well, and then place at 37°C for 2 hours to rotate and mix well;

[0064] E. After the reaction is completed, place the beads on a magnetic stand, remove the supernatant, add blocking solution to a concentration of 5 mg / mL, shake and mix, and then place at 37°C for 1 hour.

[0065] F. After blocking is completed, repeat step B and finally store the antibody-coupled magnetic beads for future use.

[0066] The filter is fixed in the middle of the first container, and its mesh diameter is 1 μm, and is used to intercept blood cells.

[0067] Step 1: Couple the anti-human IgM antibody to the magnetic bead carrier 4. According to the severity of the cold aggregation of the test sample, take an appropriate amount of magnetic beads, place them in the second container 2, and then place them on the magnetic rack, remove the supernatant and mix them evenly;

[0068] Step 2: Assemble the second container 2 containing the carrier with the first container 1, and assemble the blood collection tube containing the test sample with the connecting port 11 of the first container 1;

[0069] Step three, shake the assembled blood collection tube, first container 1, and second container 2 up and down. Shaking up and down means gently turning them upside down. You can use a laboratory shaking device or turn them upside down manually. During the inversion process, the plasma enters the second container 2 through the filter, and the cold agglutinins in the plasma bind to the anti-human IgM antibodies coated on the carrier in the second container 2. The plasma with the cold agglutinins removed passes through the filter and returns to the blood collection tube. During this process, observe the blood in the blood collection tube. When the agglutination phenomenon is resolved, it is judged that there is no obvious agglutination phenomenon.

[0070] Step 4: Place the blood collection tube at the bottom and the second container 2 at the top, so that all the plasma returns to the blood collection tube, and centrifuge instantly to return the plasma to the blood collection tube;

[0071] Step 5. Remove the blood collection tube, cover the tube cap, shake well, and perform a routine blood test.

Claims

1. A cold agglutinin removal device, characterized in that: It includes a first container, a second container, a filter, and a carrier. The first container is provided with a connection port. The interior of the first container is connected with the interior of the second container. The filter is located in the first container and is used to intercept blood cells. The second container is used to place the carrier, and the carrier is provided with anti-human IgM antibodies.

2. A cold agglutinin removal device according to claim 1, characterized in that: The mesh diameter of the filter is smaller than the diameter of the blood cells.

3. A cold agglutinin removal device according to claim 1 or 2, characterized in that: The mesh diameter of the filter is 1 μm.

4. A cold agglutinin removal device according to claim 1, characterized in that: The carrier is a magnetic bead, the diameter of the magnetic bead is larger than the diameter of the filter mesh, the anti-human IgM antibody is coupled to the magnetic bead, and the second container is detachably connected to the first container.

5. The cold agglutinin removal device according to claim 1, characterized in that: The method of coupling the magnetic beads to the anti-human IgM antibody is as follows: A. Prepare coupling solution: 100 mM MES pH 4.8, blocking solution: 3 M Ethanolamine, 1% BSA, 0.5% T-20, pH 8.0, and storage solution: 0.05% proclin-300 in PBS buffer; B. Mix the magnetic beads with a vortex oscillator, place the magnetic beads in an EP tube, add coupling solution to make the final concentration of the magnetic beads 5 mg / mL, mix with a vortex oscillator, and then place on a magnetic separation rack to enrich the magnetic beads, remove the supernatant, and then add coupling solution to repeat the washing once; C. Add coupling solution, add activator EDC and NHS to a final concentration of 1.5 mg / mL, add magnetic beads to a final concentration of 5 mg / mL. Shake and mix, then place at 37°C for 30 minutes; D. After the reaction is completed, place the mixture on a magnetic stand, remove the supernatant, and use the coupling solution to prepare the antibody so that the ratio of antibody to magnetic beads is 1:50 and the final concentration of magnetic beads is 5 mg / mL. Mix by shaking, and then place at 37°C for 2 hours. E. After the reaction is completed, place the beads on a magnetic stand, remove the supernatant, add blocking solution to a concentration of 5 mg / mL, shake and mix, and then place at 37°C for 1 hour. F. After blocking is completed, repeat step B and finally store the antibody-coupled magnetic beads for future use.

6. A cold agglutinin removal device according to claim 1, characterized in that: The carrier is a honeycomb structure or a porous structure, and the anti-human IgM antibody is coated on the carrier.

7. A cold agglutinin removal device according to claim 1 or 6, characterized in that: The carrier material is polystyrene, and the method of coating the anti-human IgM antibody on the carrier is as follows: A. Coating: Coating with anti-human IgM antibody (μ-chain specific), coating solution: pH 9.6, 0.01M carbonate buffer, incubate at 37°C for 2 hours. B. Washing: Wash with PBS buffer for 3 times and spin dry. C. Blocking: Add blocking solution and incubate at 37°C for 2 hours.

8. A cold agglutinin removal device according to claim 1, characterized in that: The first container is made of polyethylene terephthalate and / or the connection port is embedded with an inner ring made of latex.

9. The cold agglutinin removal device according to claim 1, characterized in that: The first container and / or the second container are cylindrical in shape.

10. A method for removing cold agglutinins using the cold agglutinin removal device according to any one of claims 1 to 9, characterized in that: The following steps are included: Step 1: couple the anti-human IgM antibody to the magnetic bead carrier, and according to the severity of the cold agglutination of the test sample, take an appropriate amount of magnetic beads and place them in the second container; or coat the polystyrene carrier in the second container with the anti-human IgM antibody; Step 2, assembling the second container containing the carrier with the first container, and assembling the blood collection tube containing the test sample with the connecting port of the first container; Step 3, shaking the assembled blood collection tube, the first container, and the second container up and down to make the plasma go back and forth between the blood collection tube and the second container to allow it to fully react until there is no obvious agglutination phenomenon; Step 4, placing the blood collection tube at the bottom and the second container at the top, centrifuging instantly to return the plasma to the blood collection tube; Step 5. Remove the blood collection tube, cover the tube cap, shake well, and perform a routine blood test.

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