Device for separating whole blood components

Abstract

This invention relates to a device for separating blood components (e.g., plasma or serum) in a whole blood sample. The device comprises a tube that holds 0.1-50 mL liquid volume; one or more pieces of membrane reversibly fixed inside the tube, wherein the membrane has a pore size at least 8 micrometers in diameter, and the membrane comprises an anti-red blood antibody; wherein the tube is a centrifuge or a tube adaptable to fit a centrifuge. The invention also relates to a method for separating plasma from a blood sample using the device in a low speed (300G-1000G or 300-600G) and a short time (< 3 minutes). The present device and method collect plasma from a whole blood sample in a larger volume and in a shorter time, which are useful in critical care or in emergency care.

Description

[0001] DEVICE FOR SEPARATING WHOLE BLOOD COMPONENTS

[0002] FIELD OF THE INVENTION

[0003] This invention relates to a device for separating blood components in a blood sample. The device comprises a tube that holds 0. 1-50 mL liquid volume; one or more pieces of membrane reversibly fixed inside the tube, wherein the membrane has a pore size at least 8 micrometer in diameter, and the membrane comprises an anti-red blood antibody; wherein the tube is a tube adaptable to fit a centrifuge. The invention also relates to a method for separating plasma from a blood sample using the device.

[0004] BACKGROUND OF THE INVENTION

[0005] In vitro diagnosis is a method of analyzing and diagnosing markers outside the body, which plays an important role in medical fields such as disease diagnosis, prevention, treatment, and prognosis observation. It can help doctors determine the patient's disease status, prognosis, and drug response. The in vitro diagnostic methods include biochemical diagnosis, immunodiagnosis, molecular diagnosis, blood and body fluid diagnosis, etc.

[0006] Common sample types in the field of in vitro diagnosis include blood, urine, feces, saliva, nasopharyngeal swabs, etc. Among them, blood samples are currently the most widely used and have the largest test volume. Whole blood separation is an advanced blood separation technology that scientifically separates whole blood to obtain blood products with different components to meet clinical treatment and scientific research needs. This technology is currently widely used in departments such as transfusion, hematology, and clinical laboratories in hospitals.

[0007] The principle of whole blood separation technology is based on the physical and chemical properties of different components in the blood. Whole blood is separated into different components such as red blood cells, white blood cells, platelets, and plasma through centrifugation, filtration, adsorption, and other means. Serum is the liquid that remains after the blood has clotted. Plasma is the liquid that remains when clotting is prevented with the addition of an anticoagulant. Blood components have different biological functions and clinical application values.

[0008] Red blood cells are one of the most important components of blood, responsible for transporting oxygen and nutrients. Red blood cell separation can be applied to the treatment of

[0009] 1

[0010] 184576381 1 anemia, transfusion reactions, and other diseases. White blood cells play an important role in the immune system, and white blood cell separation can be used for the treatment of immune deficiencies, infections, and other diseases. Platelets are key components for hemostasis and coagulation, and platelet separation can be used to prevent and treat diseases such as thrombosis and bleeding. Plasma contains various proteins and nutrients. Plasma separation can be used for the treatment of protein deficiency, nutritional supplementation, and other purposes.

[0011] However, whole blood separation technology has certain risks and limitations. For example, cell damage and hemolysis may occur during the separation process, affecting the quality of blood products. In in vitro diagnostic testing, the red blood cell and white blood cell layers in whole blood may interfere wi th the detection, and the proteins or enzymes released by the rupture of blood cells may have different effects on detection markers, thus seriously affecting the accuracy of test results. Therefore, for some biomarker detection in serum or plasma, pre-processing of whole blood samples is often required, i.e., separating the serum or plasma from the blood before subsequent detection.

[0012] Currently, there are mainly two types of plasma separation techniques. The first method involves centrifuging the whole blood at a high speed (e.g., 1000G) for a long time (e.g., 10 min) to achieve separation, which can be inconvenient for emergency projects and other tests that have strict requirements on testing time. The second method is the lateral flow chromatography platform, which uses filtration or adsorption methods to separate blood cells from plasma for further detection. However, this method does not have a broad application, and it provides a low throughput.

[0013] There exists a need for a device and method that quickly separate serum or plasma from a whole blood sample.

[0014] BRIEF DESCRIPTION OF THE DRAWINGS

[0015] FIG. 1 depicts a simple diagram of the device.

[0016] FIG. 2 shows the comparison of plasma separation volume by centrifugation with and without anti-RBC antibody immobilized membrane.

[0017] FIG. 3 Shows the comparison of the negative sample detection by the conventional method and the present method.

[0018] FIG. 4 shows the protocol for preparing plasma samples of Example 4.

[0019] FIG. 5 shows the correlation between positive plasma samples collected by

[0020] 2

[0021] 184576381 1 conventional method and by the present method.

[0022] DETAILED DESCRIPTION OF THE INVENTION

[0023] The present disclosure provides a device and method for separating blood components in a blood sample in a short time. The present device and method separate blood serum or plasma from a whole blood sample within 30 seconds to 3 minutes. After adding the whole blood sample to the device, the plasma or serum separation process can be completed at a lower centrifugation speed and in a shorter time, comparing to a conventional method. For example, using the present device, plasma or serum can be separated by centrifuging at 300- 1000 G for 30 seconds.

[0024] The whole blood separation device of the present invention has good test performance. Plasma or serum samples obtained after centrifuging whole blood samples in the device provide consistent test results in immunoassays comparing with the results from conventional plasma samples.

[0025] FIG. 1 depicts a simple diagram of the device.

[0026] The device comprises: (a) a tube (100) that holds a liquid sample having a volume of 0. 1-50 mL; (b) one or more pieces of membrane (101) reversibly fixed inside the tube, wherein the membrane has a pore size at least 8 micrometer in diameter, and the membrane comprises an anti-red blood cell antibody, a surfactant, and a protein to block non-specific binding of the materials in the sample to the membrane; and optionally (c) a sealing film (102) on top of the to seal the tube; wherein the tube is a centrifuge tube or a tube adaptable to fit into a centrifuge for centrifugation.

[0027] The liquid sample in general has a volume of 0.1-50 mL, 0.1-10 mL, 0.1-1 mL. 0.2-50 mL, or 0.2-15 m, 0.2-10 mL, or 0.2-5 mL.

[0028] The tube may be made of plastic, glass, metal. Teflon, or ceramic.

[0029] The membrane is made of one or more materials selected from the group consisting of: glass fiber, cotton linter, filter paper, a polyamide composite, an acetate fiber, nitrocellulose, cellulose acetate, polyethersulfone, polysulfone, polyacrylonitrile, polyamide, a cellulose acetate-nitrocellulose composite, and polycarbonate. Cotton linters are the short, thick-walled, curly fibers that remain on cotton seeds after the cotton fibers are removed through ginning.

[0030] 3

[0031] 184576381 1 The membrane is reversibly atached or fixed in the tube at a position < 50% height of the tube. For example, it is close to the botom of the tube. The membrane is not close to the top of the tube such that when the membrane will not be damaged when the blood is added to the tube. For example, when a seal film is punctured to apply blood to the tube, the membrane will not be damaged. The membrane is reversibly or loosely fixed in the tube, which means that the membrane is not permanently fixed to the tube. When the seal is broken and a blood sample is added to the tube, and when the tube is centrifuged, the membrane will move toward to the botom of the tube.

[0032] In one of the embodiments, the volume ratio of the whole blood sample to the total blood filtration membrane is 10-50 pL / mm3. Each membrane volume is its thickness times area. The total membrane volume is the addition of each membrane volume.

[0033] The membrane has a pore size at least the same as. or greater than the size of red blood cell to allow the binding and / or passage of red blood cell toward the botom of the tube. In one embodiment, the pore size has a diameter of at least 8p, or at least 9p.

[0034] The membrane is immobilized with an anti-human red blood cell antibody to bind the red blood cell onto the membrane. The membrane is pre-treated and dried with anti-human red blood cell antibodies in a buffer solution with pH 4-10. The buffer for example is PBS, MES, TRIS, and HEPES.

[0035] The present invention uses one or more membranes bound with an anti-human red blood cell antibody in centrifuging whole blood cells. During centrifugation, the red cell bound-membranes move to the botom of the cells. When membranes bound with an antihuman red blood cell antibody are used in centrifugation, red blood cells setle to the botom of the tube faster and the cells are more compacted, which leads to collecting higher liquid volume of plasma or serum. Obtaining a higher volume of plasma or serum from whole blood is important, in particular for pediatric patients because of the difficulty to obtain a large volume of whole blood from these patients.

[0036] In one embodiment, the membrane is pre-treated and dried with a surfactant to accelerate blood filtration, where the surfactant includes a cationic, anionic, zwiterionic, or nonionic surfactant. The nonionic surfactant includes Tween and Triton.

[0037] In one embodiment, the membrane further comprises a preservative or an antimicrobial agent to prevent the growth of microorganisms. For example, the preservative

[0038] 4

[0039] 184576381.1 includes ProCiin 300 (a viscous solution containing 93-95% modified glycol and 2-3% alkyl carboxylate), azide, etc.

[0040] In one embodiment, the device comprises a centrifuge sleeve to adapt the tube to a centrifuge, such as an angle rotor centrifuge or a horizontal rotor centrifuge.

[0041] The sealing film on top of the plastic tube seals the tube. The sealing film can be made of aluminum foil, paper, or plastic. The seal should have the property of easily puncturable and removable such that a blood sample can be delivered without breaking pieces of the seal into the tube.

[0042] In one embodiment, the tube is heat-sealed with an aluminum film to maintain the dryness of the membrane(s) in the tube.

[0043] In operation, the seal of the tube is punctured, for example, with a needle, and then the blood is added to the tube.

[0044] The present invention also provides a method for obtaining plasma or serum from a blood sample. The method comprises the steps of: adding a whole blood sample into a tube, wherein the tube comprises one or more pieces of membrane reversible attached inside the tube, wherein the membrane has a pore size at least 8 micrometer in diameter, and the membrane comprises an anti-red blood antibody, a surfactant, and a protein to block nonspecific binding in the sample; centrifuging the tube at 300-1000G for 20 second to 3 minutes to separate plasma or serum from the blood; and removing the plasma or serum from the blood.

[0045] The whold blood sample in general has a volume of 0.1-50 mL, 0.1-10 mL, 0.1-1 mL. 0.2-50 mL, or 0.2-15 m, 0.2-10 mL, or 0.2-5 mL.

[0046] In one embodiment, the tube is centrifuged for 300-800G.

[0047] In another embodiment, the tube is centrifuged for 300-600G.

[0048] In a preferred method, the centrifuging is 20 seconds to 1 minute.

[0049] Currently, the separation of plasma from conventional whole blood samples requires high-speed centrifugation at 1000G for 10 minutes to obtain plasma samples for detection and analysis. The present device and method can quickly complete the filtration and plasma separation of a whole blood sample in a short time, for example, 30 seconds at a low speed of 300G, through the combined actions of antigen-antibody binding on the membrane, membrane filtration, and centrifugal force. The present device and method provide rapid separation of whole blood sample and only requires a low-speed centrifuge. The present

[0050] 5

[0051] 184576381 1 device and method provide a high speed to collect a plasma sample, which is critical in emergency care and critical care.

[0052] The test results of the plasma samples obtained by the present device and method are consistent with the test results of plasma samples obtained by a conventional method.

[0053] The invention is illustrated further by the following examples that are not to be construed as limiting the invention in scope to the specific procedures described in them.

[0054] EXAMPLES

[0055] Example 1. Preparing anti-red blood cell antibody coated membrane

[0056] A blood filtration membrane contained mixed materials of cotton linter and glass fiber was pretreated as follows. The membrane with a size of 20 cm x 30 cm was laid flat on a screen window, and 40 mL of a treatment solution (0.05M phosphate buffer, pH 6.0, 0.6% Tween-20, 0.4% BSA, 0.02 pg / mL mouse anti human-red blood cell antibody (Changzhou Geling Biotechnology7, Co., Ltd), 0.02% Proclin300) was evenly spread over the membrane, and then the membrane was left air dried overnight. The dried membrane was cut into squares of 2.5 mm * 2.5 mm, and six pieces of the membrane were used in the 350 pL centrifuge tube.

[0057] Example 2. Comparing plasma volumes collected by present method vs. same method without membrane

[0058] One set of 10 plasma samples were prepared from 10 whole blood samples, by using the present method of adding 300 pL of each whole blood sample in a 350 pL non- conventional centrifuge tube containing the 6 pieces of membrane as described in Example 1, and then centrifuged at 300xG for 30 seconds.

[0059] Another set of 10 plasma samples were prepared from the same 10 whole blood samples as above, by using the present method of adding 300 pL of each whole blood sample in a 350 pL non-conventional centrifuge tube but without membrane, and then centrifuged at 300xG for 30 seconds. The collected volume of each sample was measure, and the mean plasma volumes obtained by the present method vs. the same method without membrane were compared and shown in FIG. 2. The results show7that the mean plasma collected by the

[0060] 6

[0061] 184576381 .1 present method is 110 pL. which is about 40% higher than the 76 pL collected by the method without membranes.

[0062] Example 3. Comparing detection of negative samples collected by present method vs. conventional method

[0063] Ten negative plasma samples prepared by the conventional method (centrifuged at 1000G for 10 minutes) and by the present method (centrifuged at 300G for 30 seconds, with membranes) were tested by Anti-Mullerian Hormone (AMH) Assay Kit (ET healthcare, Shuzhou, China), which is a fluorescence immunoassay. The results were shown below in FIG. 3. The results show that the negative plasma samples collected by conventional method or by the present method both showed negative results in AMH test.

[0064] Example 4. Comparing detection of positive samples collected by present method vs. conventional method

[0065] Each of the negative plasma of whole blood samples with negative AMH was collected by conventional centrifugation method (1000G, 10 minutes). The collected negative plasma samples were then pooled together as a negative plasma pool.

[0066] The high positive plasma of a whole blood sample having a high positive AMH value was collected by conventional centrifugation method (1000G, 10 minutes).

[0067] The high positive plasma was then serially diluted (1:3 each dilution) into the negative plasma pool to prepare 5 positive AMH plasma samples with different amounts of AMH. Then tw o sets of the 5 positive plasma samples were each mixed with 150 pLof blood cells. One set of the mixtures w as centrifuged by the conventional method (1000G, 10 minutes), the other set of the mixtures was centrifuged by the present method (300G, 30 seconds), to prepare two sets of plasma samples. The plasma sample preparation protocols are shown in FIG. 4.

[0068] Each plasma sample was then tested by the same AMH test as described in Example 3. The correlation of plasmas collected by the present method (300G, 30 seconds) vs. a conventional method (1000G, 10 minutes) is shown in FIG. 5. The results show' a good correlation coefficient with R2= 0.9994.

[0069] The inventions, and the manner and process of making and using them, are now

[0070] 7

[0071] 184576381 1 described in such full, clear, concise and exact terms as to enable any person skilled in the art to which they pertain, to make and use the same. It is to be understood that the foregoing describes preferred embodiments of the present disclosure and that modifications may be made therein without departing from the scope of the present disclosure as set forth in the claims.

[0072] 8

[0073] 184576381 .1

Claims

WHAT IS CLAIMED IS:

1. A device for separating blood components in a blood sample, comprising:(a) a tube that holds 0. 1-50 mL liquid volume, and(b) one or more pieces of membrane reversibly fixed inside the tube, wherein the membrane has a pore size at least 8 micrometer in diameter, and the membrane comprises an anti-red blood cell antibody; wherein the tube is a centrifuge tube or a tube adaptable to fit into a centrifuge for centrifugation.

2. The device of claim 1 , wherein the membrane is made of a material selected from the group consisting of: glass fiber, cotton linter, a polyamide composite, an acetate fiber, nitrocellulose membrane, cellulose acetate, a poly ethersulfone, a poly sulfone. polyacrylonitrile, polyamide, a cellulose acetate-nitrocellulose composite, and polycarbonate.

3. The device of claim 1, wherein the membrane has a pore size at least 9 micrometer in diameter.

4. The device of any one of claims 1-3, wherein the membrane further comprises a surfactant, and a protein to block non-specific binding of the materials in the sample to the membrane.

5. The device of claim 4, wherein the membrane further comprises a preservative.

6. The device of any one of claims 1-3, further comprises a sealing film on top of the tube to seal the tube.

7. The device of claim 6, wherein the sealing film is made of aluminum foil, paper, or plastic.

8. The device of claim 7, wherein the sealing film is puncturable.9184576381 19. The device of any one of claims 1-3, wherein the tube is made of plastic, glass, metal, Teflon, or ceramic.

10. The device of any one of claims 1-3, wherein the tube holds 0.2-50 mL liquid volume11. A method for separating plasma or serum from a blood sample, comprising: obtaining the device of claim 1, adding 0.1-50 mL of a blood sample into the tube, centrifuging the tube at 300-1000G for 20 second to 3 minutes to separate plasma or serum from the blood; and removing the plasma from the blood.

12. The method of claim 11, wherein the centrifuging time is 20 seconds to 1 minute.

13. The method of claim 11, wherein the tube is centrifuged at 300-600G.

14. The method of any one of claims 11-13, wherein 0.2-50 mL of the blood sample is added to the tube.10184576381 .1

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