Separation device, method of blood separation and use

Abstract

Embodiments relate to a separation device, a method of blood separation and use. The separation device comprises a tube, a valve and a separation gel. The tube has a third tube section in the middle, dividing the tube into a first tube section and a second tube section. The first tube section has an opening and the second tube section is a closed cavity. The valve comprises a valve cover facing the first tube section and an elastic body connected thereto. The valve cover has an outer diameter between the inner diameter of the first tube section and the third tube section to be positioned at the third tube section during centrifugation. The valve cover has a gap portion to form a gap flow channel when cooperating with the third tube section to allow blood and separation gel to pass through during centrifugation and complete the layering. The elastic body springs back after centrifugation to lock the valve at the third tube section to mechanically separate the plasma from the blood cells. The separation gel has a density between the plasma and the blood cells and is positioned at the interface between the plasma and the blood cells after centrifugation. Thus, the separation of the plasma from the blood cells in the separation device and the separation thereof can be achieved after one centrifugation.

Description

Technical Field

[0001] This application relates to the field of in vitro diagnostic sample processing technology, and in particular to a separation device and method for separating and isolating plasma from whole blood samples. Specifically, it relates to a separation device that includes a third tube section and a valve body, and is equipped with a separation gel, so that after one centrifugation of whole blood, plasma and blood cells are separated into layers and mechanically isolated after centrifugation. Background Technology

[0002] In the field of biology, blood separation technology plays a vital role in clinical diagnosis, biomedical research, and personalized medicine. Blood plasma contains a large number of proteins, hormones, free nucleic acids, and metabolites essential for clinical and research purposes, forming a crucial foundation for key medical activities such as disease diagnosis, biomarker analysis, and gene sequencing. Efficient and pollution-free blood separation technology directly impacts diagnostic accuracy and the precision of experimental results.

[0003] Currently, the most widely used blood separation methods in clinical and laboratory settings rely primarily on traditional centrifugation equipment. While centrifugation offers high separation efficiency, it also has significant limitations. Therefore, there is still considerable room for improvement to achieve efficient and convenient separation of plasma and blood cells. Summary of the Invention

[0004] In a first aspect of an exemplary embodiment, a separation device is provided. The separation device includes: a tube body comprising: a first tube segment including an opening; and a second tube segment including a bottom and communicating with the first tube segment via a third tube segment, wherein the radial dimension of the third tube segment is smaller than that of the first tube segment and the second tube segment; and a valve body comprising: a valve cover having a maximum radial dimension smaller than that of the first tube segment and larger than that of the third tube segment, such that the valve cover is movable within the first tube segment in a first direction from the opening to the bottom and positioned at the third tube segment under centrifugal force; a spacer portion disposed on the bottom surface of the valve cover facing the second tube segment and protruding along the first direction, the end of the spacer portion away from the valve cover abutting against the third tube segment; and an elastomer connected to the side of the valve cover near the second tube segment.

[0005] In a second aspect of an exemplary embodiment, a method for blood separation is provided. The method includes: placing blood into a tube of a separation device according to the first aspect; centrifuging such that a valve body is positioned in the third tube segment under centrifugal force and locked in the third tube segment after centrifugation, thereby placing plasma of the blood in the first tube segment and mechanically isolating it from blood cells of the blood located in the second tube segment; and obtaining the plasma from the first tube segment.

[0006] In a third aspect of the exemplary embodiments, an application is provided for the use of the separation apparatus according to the first aspect of the exemplary embodiments in the preparation of samples of circulating free deoxyribonucleic acid (cfDNA).

[0007] It should be understood that the description in the Summary of the Invention is not intended to limit the key or essential features of the exemplary embodiments, nor is it intended to restrict the scope of the exemplary embodiments. Other features of the exemplary embodiments will become readily apparent from the following description. Attached Figure Description

[0008] To better understand the above and other objects, features, advantages, and functions of the exemplary embodiments, reference can be made to the preferred embodiments shown in the accompanying drawings. Like reference numerals in the drawings refer to like parts. Those skilled in the art should understand that the drawings are intended to schematically illustrate preferred embodiments of the exemplary embodiments and are not intended to limit the scope of the exemplary embodiments; the various parts in the drawings are not drawn to scale.

[0009] Figure 1 A schematic diagram of the structure of an example separation device according to an exemplary embodiment is shown;

[0010] Figures 2A to 2C A schematic diagram showing the relative positions of the valve body and the tube body, and the blood stratification, is shown in the separation device in the non-centrifuged, centrifuged, and centrifuged states.

[0011] Figures 3A to 3B It shows Figures 2A to 2C A partial enlarged view of the mating area between the valve body and the third pipe section; and

[0012] Figures 4 to 7 A schematic diagram of comparative experimental results used to characterize plasma recovery, hemolysis level, DNA fragment distribution, and methylation consistency is shown. Detailed Implementation

[0013] Various embodiments will now be described with reference to the accompanying drawings, wherein similar reference numerals are used throughout to denote similar elements. In the following description, numerous specific details are set forth for purposes of explanation in order to facilitate a thorough understanding of one or more embodiments. However, it may be apparent in some or all cases that any of the embodiments described below can be practiced without employing the specific design details described below. In other instances, well-known structures and devices are illustrated in block diagram form to facilitate the description of one or more embodiments. A simplified overview of one or more embodiments is given below to provide a basic understanding of the embodiments. This overview is not an exhaustive summary of all contemplated embodiments, is not intended to identify key or essential elements of all embodiments, nor is it intended to define the scope of any or all embodiments.

[0014] References to “embodiment” or “one embodiment” within the framework of this description are intended to indicate that a particular configuration, structure, or feature described with respect to an embodiment is included in at least one embodiment. Therefore, phrases such as “in an embodiment” or “in one embodiment” that may appear at one or more points in this description do not necessarily refer to the same embodiment. Furthermore, in one or more embodiments, particular constructions, structures, or features may be combined in any suitable manner.

[0015] In the following disclosure, unless otherwise indicated, references to absolute positional modifiers (such as the terms “front,” “back,” “top,” “bottom,” “left,” “right,” etc.) or relative positional modifiers (such as the terms “above,” “below,” “higher,” “lower,” etc.), or references to orientational modifiers (such as “horizontal,” “vertical,” etc.), refer to the orientation shown in the figure.

[0016] As discussed above, in recent years, with the rapid development of precision medicine, higher requirements have been placed on the purity and efficiency of plasma component separation in blood samples. This is especially true in applications highly sensitive to sample quality, such as cfDNA (Cell-Free DNA) detection, where the limitations of traditional separation devices and methods are becoming increasingly apparent. The gel barrier formed in traditional separation gel blood collection tubes after centrifugation is easily affected by factors such as transport vibration and temperature fluctuations, leading to backmixing of plasma and blood cells. This results in gDNA (Genomic DNA) released from ruptured nucleated cells contaminating cfDNA, thus affecting the accuracy of test results. Furthermore, blood collection tubes relying on chemical stabilizers may introduce cross-linking or immobilization effects due to their preservatives, potentially interfering with downstream detections such as methylation, thus limiting the compatibility and application scope of the tests. Simultaneously, the traditional process requires opening the cap to transfer plasma after centrifugation, which is not only cumbersome and inefficient but also poses risks of biocontamination, aerosol exposure, and leakage, making it difficult to execute with high quality in blood collection or primary healthcare settings. In addition, some mechanically separated blood collection tubes have high requirements for centrifugation conditions and have problems such as unstable plasma recovery rate and easy locking into the lower cavity, resulting in waste, which cannot meet the urgent clinical needs for rapid, efficient and low-pollution blood separation.

[0017] In view of this, the exemplary embodiment proposes a separation device with novel structural design, stable separation effect, convenient operation and strong compatibility. Through the special segmented structure of the tube body 11, the three-state switching function of the valve body 12 and the synergistic effect of the separation gel 23, the efficient separation and reliable isolation of plasma and blood cells can be completed in one low-speed centrifugation, effectively solving many shortcomings of traditional technology.

[0018] The following will combine Figures 1 to 3BThe scheme according to the exemplary embodiment will be described in detail below. First, the structure and connection relationship of each component of the separation device 1 will be described in detail. (Refer to...) Figure 1 The diagram shows an example separation device 1 according to an exemplary embodiment. Figures 2A to 2C A schematic diagram of an example blood separation process according to an exemplary embodiment is shown, wherein... Figure 2A In the uncentrifuged state, Figure 2B This represents the state of the centrifugation process. Figure 2C This is the state after centrifugation. The tube body 11, as the basic supporting component of the entire device, includes a first tube section 111, a second tube section 112, and a third tube section 113 connecting the two. The tube opening 115 is located at the top of the first tube section 111 for the blood sample 20 collected from the experimenter to enter. The tube bottom 116 is located at the bottom of the second tube section 112, forming a closed cavity to ensure that the blood sample does not leak during separation. Figure 2A As shown, the blood 20 collected from the experimenter, when not subjected to centrifugal force, comprises plasma 21 and blood cells 22 mixed together, wherein the blood cells 22 mainly include red blood cells 221 and white blood cells 222. The purpose of this embodiment is to separate the plasma 21 from the blood cells 22, thereby enabling the plasma 21 to be extracted for subsequent processing.

[0019] like Figure 1 As shown, the radial dimensions of the first tube segment 111 provide sufficient space for the initial containment of blood samples and for stratification during centrifugation. The radial dimensions of the second tube segment 112 are similar to those of the first tube segment 111, as shown... Figure 2A As shown, when not subjected to centrifugal force, the second tube segment 112 can accommodate a suitable volume of separating gel 23, the function of which will be described in detail below. The third tube segment 113 is a reduced section of the tube body 11, with a radial dimension smaller than that of the first tube segment 111 and the second tube segment 112. The third tube segment 113 serves as the positioning and locking interface of the valve body 12, playing a crucial role in the blood separation process. Figure 1 As shown, the third pipe section 113 further includes a first reduction section 1131 and a second reduction section 1132. The radial dimension of the first reduction section 1131 gradually decreases along a first direction D1 (i.e., from the pipe opening 115 to the pipe bottom 116), eventually forming the narrowest section 1133. The first direction D1 is defined as the direction from the pipe opening 115 to the pipe bottom 116. The radial dimension of the second reduction section 1132 gradually decreases along a second direction D2 (opposite to the first direction D1), i.e., from the pipe bottom 116 to the pipe opening 115, from the second pipe section 112 to the narrowest section 1133. The second direction D2 is opposite to the first direction D1, i.e., from the pipe bottom 116 to the pipe opening 115. This bidirectional reduction structural design is used to form a valve body locking interface after centrifugation / facilitates valve body positioning, which will be described in detail below.

[0020] It should be noted that the first reduction section 1131 and the second reduction section 1132 can be designed as a cone shape, a single step, a multi-step or a multi-stage contraction section according to actual application requirements. For example, in this embodiment, the first reduction section 1131 adopts a cone shape and the second reduction section 1132 adopts a single step structure, which not only ensures structural strength but also optimizes the blood flow path.

[0021] The valve body 12 is the core component for blood separation and isolation. It is integrally housed within the first section 111 of the tube body 11, allowing for displacement and state changes along the first direction D1 and the second direction D2. The valve body 12 includes a valve cover 121, a spacer 122, and an elastomer 123, all connected as a single unit via a rubber-coated integral molding process, ensuring structural stability and coordinated movement. The valve cover 121 is rigid and can be made of rigid materials such as polypropylene (PP) or polycarbonate (PC). Its maximum radial dimension is smaller than that of the first section 111, allowing the valve cover 121 to move freely within the first section 111 along either the first direction D1 or the second direction D2. Simultaneously, its maximum radial dimension is greater than that of the third section 113 (i.e., the radial dimension of the narrowest part 1133), enabling it to be stopped and positioned by the third section 113 during centrifugation, preventing the entire valve body 12 from falling into the second section 112.

[0022] Figures 3A to 3B A partially enlarged view of an example blood separation process according to an exemplary embodiment is shown, wherein Figure 3A This represents the state of the centrifugation process. Figure 3B This is the state after centrifugation. (Reference) Figure 3B The valve cover 121 further includes a middle part 1211 and a circumferential part 1212. The longitudinal dimension of the middle part 1211 along the first direction D1 is larger than the longitudinal dimension of the circumferential part 1212 along the first direction D1, forming a "high in the middle and low around the periphery" flow guide surface structure. This structural design can effectively reduce the accumulation of blood cells 22 above the valve cover 121 during centrifugation, promote the blood cells 22 to slide down along the flow guide surface and enter the second tube section 112 through the third tube section 113, and ensure the smooth progress of the stratification process.

[0023] refer to Figure 3BThe spacer portion 122 protrudes from the side of the valve cover 121 facing the second pipe section 112. Its specific structure can be multiple protrusions, ribs, support feet, a grid support structure, or annular flanges, as long as it can form a gap flow channel when the valve cover 121 abuts against the third pipe section 113. In an embodiment, the spacer portion 122 can adopt an annular rib structure, evenly distributed along the circumference of the valve cover 121. When the valve cover 121 abuts against the first narrowing portion 1131 of the third pipe section 113 under centrifugal force, the end of the annular rib away from the valve cover 122 contacts the inner wall of the third pipe section 113, forming an annular gap flow channel between the valve cover 121 and the third pipe section 113. This flow channel provides a passage for the relative flow of blood 20 and / or separating gel 23, ensuring that the components of blood 20 can pass smoothly during centrifugation and avoiding flow obstruction or stratification interruption due to the complete adhesion between the valve cover 121 and the third pipe section 113.

[0024] The elastomer 123 is made of materials with good elasticity and resilience, such as silicone rubber and thermoplastic elastomer (TPE). Its upper end is fixed to the side of the valve cover 121 near the second pipe section 112, and its lower end forms a deformable locking part. This ensures both the tensile deformation capability during centrifugation and the rebound locking effect after centrifugation. The design of the elastomer 123 is key to achieving the three-state switching of the valve body 12. Its morphological changes in different states directly determine the workflow of the separation device 1: In the non-centrifugation state, the elastomer 123 is in the first naturally relaxed state, and the whole body is in a contracted state. At this time, the valve body 12 is located in the upper region of the first tube section 111, and sufficient flow space is maintained between it and the inner wall of the tube body 11, which facilitates the smooth entry of blood samples into the tube body 11 during blood collection. It also allows some blood and separating gel 23 to pass through the valve body 12 and the third tube section 113 into the second tube section 112, laying the spatial distribution foundation for subsequent centrifugation and stratification. When the separation device 1 is in the centrifugation state, under the action of centrifugal force, the elastomer 123 undergoes axial tensile deformation, and its equivalent lateral dimension decreases, allowing it to partially extend into the third tube section 113 and the second tube section 112. At this time, the valve cover 121 abuts against the first reduced part 1 of the third tube section 113 under the pressure of centrifugal force and blood components. 131. The gap flow channel formed by the partition 122 ensures the continuous flow of blood components, causing blood cells 22 to continuously sink to the second tube section 112 under the action of centrifugal force, while plasma 21 accumulates upward in the first tube section 111. The separating gel 23 gradually migrates to the interface between plasma 21 and blood cells 22 under the action of density difference. After centrifugation, the separation device 1 is in a centrifuged state, the centrifugal force disappears, and the elastic body 123 rebounds and contracts under the action of its own elastic recovery force. The outer periphery of its circumferential skirt forms a tight radial compression fit and / or interference fit with the inner wall of the narrowest part 1133 of the third tube section 113, thereby fixing the valve body 12 at the third tube section 113 and blocking the fluid communication between the first tube section 111 and the second tube section 112, realizing the mechanical isolation between the first tube section 111 and the second tube section 112, so that the plasma 21 in the upper part is completely isolated from the blood cells 22 and part of the separating gel 23 in the lower part, avoiding backmixing and contamination during transportation or subsequent operations.

[0025] To ensure the correct installation orientation of the valve body 12 within the pipe body 11 and to avoid inverted assembly affecting performance, the longitudinal dimension of the valve body 12 along the first direction D1 is greater than the radial dimension of the first pipe section 111 and the second pipe section 112, and also greater than the radial dimension of the third pipe section 113. This dimensional design enables the valve body 12 to maintain its orientation after assembly, ensuring that it can be stably placed within the pipe body 11 with the valve cover 121 facing upwards and the elastic body 123 facing downwards, effectively reducing the risk of assembly errors.

[0026] Separating gel 23, serving as an auxiliary separation and barrier component, is filled within tube 11. Its density is between that of plasma 21 and blood cells 22, preferably in the range of 1.029–1.077 g / mL, more preferably 1.03–1.07 g / mL, and even more preferably 1.04–1.06 g / mL. This density design ensures that under centrifugal force, separating gel 23 can be precisely positioned at the interface between plasma 21 and blood cells 22, forming a stable physical barrier. This barrier, in conjunction with the mechanical isolation of valve body 12, further reduces the risk of backmixing. The pre-positioned location of separating gel 23 can be near the third tube section 113 or within the second tube section 112. In this embodiment, separating gel 23 is pre-positioned in the area of ​​the second tube section 112 near the third tube section 113, so that it can quickly migrate to the interface during centrifugation. Furthermore, there is a strict matching relationship between the volume of the separating gel 23 and the volume of the tube 11, specifically satisfying one of the following two conditions: first, the total volume of the separating gel 23 is not less than the volume of the second tube segment 112; second, after centrifugation, the volume of the blood cells 22 and the volume of the separating gel 23 that moves down into the second tube segment 112 are not less than the volume of the second tube segment 112. The core purpose of this volume matching design is to ensure that the second tube segment 112 is mainly occupied by blood cells 22 and separating gel 23 after centrifugation, avoiding the plasma 21 from being locked by the valve body 12 after entering the second tube segment 112 and unable to be recovered, thereby ensuring a high recovery rate and consistency of plasma samples, and even in scenarios with low blood volume or low hematocrit (HCT), a sufficient amount of plasma for testing can be stably obtained.

[0027] The cap 13, as a sealing component, is securely fixed at the tube opening 115 to maintain a negative pressure environment inside the tube body 11, allowing blood samples to be drawn into the tube via vacuum blood collection. It also prevents sample leakage and external contamination during centrifugation, transportation, and storage. The cap 13 features a puncture-compatible structure, compatible with standard vacuum blood collection needles, ensuring both convenience and a tight seal during the blood collection process.

[0028] In addition to the main components mentioned above, the separation device 1 also includes an anticoagulant and a nuclease inhibitor to ensure the stability of blood samples and the reliability of subsequent testing. The anticoagulant can be one or more of K2EDTA (dipotassium EDTA), K3EDTA (tripotassium EDTA), sodium citrate, heparin salt, sodium fluoride, ACD (citric acid-glucose anticoagulant), and CTAD (sodium citrate-theophylline-adenosine-dapamide complex anticoagulant). The nuclease inhibitor can be one or more of EDTA, ATA (oligocarboxylic acid), vanadate-ribonucleoside complex, dithiothreitol (DTT), and TCEP (tris(2-carboxyethyl)phosphine). These reagents can be loaded via tube wall coating (dry powder / film), pre-prepared solution, or a reagent layer placed in the adjacent area of ​​the separating gel 23. In this embodiment, a dry powder coating is used on the tube wall, allowing the reagents to quickly contact and dissolve in whole blood after blood collection, rapidly exerting their anticoagulant effect and inhibiting nuclease activity, thus preventing blood coagulation and cfDNA degradation.

[0029] Next, the assembly process of the separation device 1 is described in detail, and the specific steps are as follows: First, according to the preset formula and dosage, the anticoagulant and nuclease inhibitor are evenly coated on the inner wall of the tube 11 to form a dry powder coating, ensuring that the reagents can dissolve quickly after blood collection; Second, a quantitative amount of separating gel 23 is injected into the area of ​​the second tube section 112 near the third tube section 113 of the tube 11, ensuring that the preset position of the separating gel 23 is accurate and avoiding the impact of positional deviation on the interface positioning after centrifugation; Third, the valve body 12 is connected from the tube The valve body 12 is inserted into the first section 111 of the tube body 11 through port 115, ensuring the correct installation direction, i.e., valve cap 121 facing upwards and elastic body 123 facing downwards. Initially, the valve body 12 is located at the upper part of the first section 111 and will not fall into the second section 112 due to the limiting effect of the third section 113. Fourthly, a vacuum is drawn inside the tube body 11 using specialized equipment to create a stable negative pressure environment. Then, the cap 13 is installed at the port 115 and sealed, completing the assembly of the entire separation device 1. Through the above assembly process, the separation device 1 possesses complete functions of vacuum blood collection, single-stage centrifugation and isolation, and direct sampling, with a stable structure and reliable performance.

[0030] The blood separation method is described in detail below, based on the separation device 1 described above. In general, the blood separation method includes placing blood 20 into the tube 11 of the separation device 1 described above; centrifuging, causing the valve body 12 to be positioned in the third tube segment 113 under centrifugal force and locked in the third tube segment 113 after centrifugation, thereby placing the plasma 21 of the blood 20 in the first tube segment 111 and mechanically isolating it from the blood cells 22 of the blood 20 located in the second tube segment 112; and obtaining the plasma 21 from the first tube segment 111. Specifically, the blood separation method includes steps such as blood collection and pretreatment, one low-speed centrifugation for stratification, locking and confirmation after centrifugation, preservation / transportation, and plasma sampling, each step corresponding to the three-state switching of the valve body 12. According to this exemplary blood separation method, automatic locking and isolation after centrifugation can be achieved with only one centrifugation process, reducing the need for tube transfer and secondary centrifugation.

[0031] The first step is blood collection and pretreatment. First, the assembled separation device 1 is removed. At this time, the valve body 12 is in a relaxed state before centrifugation. Figure 2A As shown, the elastomer 123 is in its first state of natural contraction, and the valve body 12 is located at the upper part of the first tube segment 111, maintaining a flow space between it and the inner wall of the tube body 11. Using a vacuum blood collection method, the blood collection needle punctures the puncture site of the tube cap 13. Under the negative pressure inside the tube body 11, whole blood 20 enters the first tube segment 111 through the tube opening 115 and comes into contact with the anticoagulant and nuclease inhibitor on the inner wall of the tube. The reagents dissolve rapidly and take effect, preventing blood coagulation and cfDNA degradation.

[0032] The second step is to apply centrifugal force. (Corresponding to...) Figure 3A and Figure 2B The state shown is as follows. The pretreated separation device 1 is placed in a centrifuge. During centrifugation, under the action of centrifugal force, the valve body 12 changes from a relaxed state to a stretched state: the valve cover 121 moves along the first direction D1 under the action of centrifugal force and the pressure of blood components, and finally the spacer portion 122 of the valve body 12 abuts against the first reduced portion 1131 of the third tube section 113 and is locked in place; the elastic body 123 is stretched and deformed axially under the action of centrifugal force, its equivalent lateral dimension decreases, and the lower end of the spherical crown and the circumferential skirt structure extend into the third tube section 113 and the second tube section 112; simultaneously, the spacer portion 122 on the valve cover 121 contacts the inner wall of the third tube section 113, forming a stable gap flow channel to ensure continuous flow of blood components. Under the combined action of centrifugal force and density difference, the components in the blood 20 stratify: the blood cells 22 with the highest density overcome resistance under the action of centrifugal force, migrate downwards through the gap flow channel, and finally deposit at the bottom of the second tube section 112, as shown. Figure 2BAs shown, white blood cells are represented by 222, and red blood cells by 221. Plasma 21, with the lowest density, accumulates upwards, occupying most of the space in the first tube segment 111. The separating gel 23, with a density between plasma 21 and blood cells 22, gradually migrates towards the interface between plasma 21 and blood cells 22 under the combined action of centrifugal force and buoyancy, eventually settling at this interface to form a stable physical barrier, initially blocking contact between plasma 21 and blood cells 22, thereby achieving separation of plasma 21 and blood cells 22. In one implementation, the relative centrifugal force for a single centrifugation can be no greater than 3000g and the centrifugation time no greater than 10 minutes. This means that even under relatively low relative centrifugal force, separation of plasma 21 and blood cells 22 can be achieved.

[0033] The third step is to lock, isolate, and confirm the centrifugation process. Figure 2C The state is shown. After centrifugation, as the centrifugal force disappears, the elastomer 123 rebounds and contracts from its stretched state under its own elastic restoring force. The outer periphery of its circumferential skirt fits tightly against the inner wall of the narrowest part 1133 of the third tube section 113, forming a radial compression fit, thereby firmly locking the valve body 12 at the third tube section 113, achieving the isolation between the first tube section 111 and the second tube section 112. At this time, a clear three-layer structure is formed in the separation device 1: the first tube section 111 contains a plasma layer 21, with a barrier layer formed by the separating gel 23 in the middle; the second tube section 112 contains a blood cell layer 22. The plasma layer 21 and the blood cell layer 22 are doubly isolated through the physical isolation of the valve body 12 and the barrier effect of the separating gel 23, effectively preventing backmixing during transportation or subsequent operations.

[0034] The fourth step is preservation / transportation and plasma sampling. After centrifugation and locking, the separation device 1 can be preserved or transported in a vertical position without special temperature control or shockproof packaging. Due to the mechanical isolation effect of the valve body 12, even if temperature fluctuations or slight shaking occur during transportation, even if a small number of blood cells 22 rupture, the released gDNA and other products are unlikely to cross the barrier and enter the plasma 21 layer, effectively ensuring the quality stability of the plasma sample. When downstream testing is required, after confirming that the valve body 12 is in a stable and locked state, open the tube cap 13 and use a pipette to directly aspirate the plasma 21 sample from the upper part of the first tube segment 111. When aspirating, the pipette tip should be placed in the upper middle part of the plasma 21 layer to avoid touching the interface of the separating gel 23 and the valve cap 121 of the valve body 12, so as to reduce the risk of the separating gel 23 or blood cell 22 components being mixed into the plasma sample. This method eliminates the need for additional operations such as opening the cap, transferring the sample to a tube, or secondary centrifugation, allowing for the acquisition of high-purity plasma samples. It not only simplifies the operational process and improves work efficiency but also reduces the risks of biological contamination, aerosol exposure, and sample leakage. It is particularly suitable for blood collection sites, primary healthcare settings, or large-scale sample processing scenarios. Example

[0035] To verify the performance advantages of the separation device 1 in the exemplary embodiment, a series of comparative experiments were conducted, and the experimental results are as follows: Figures 4 to 7 As shown below, the experimental design, experimental procedure, and results analysis are described in detail.

[0036] The first step was a comparative experiment on plasma recovery rates to verify the advantages of the device of the present invention in terms of plasma recovery rate. Ten healthy volunteers were selected as the subjects of the experiment. A paired sampling method was used. Each volunteer collected one tube of whole blood using the separation device 1 of the present invention (referred to as the Example), conventional EDTA anticoagulant blood collection tubes (BD Vacutainer® EDTA Tubes, referred to as Comparative Example 1), plasma separating gel blood collection tubes (BD Vacutainer® PPT™, referred to as Comparative Example 2), mechanically separated plasma blood collection tubes (BD Vacutainer Barricor, referred to as Comparative Example 3), and cell-free DNA preservation blood collection tubes (Streck blood collection tubes, referred to as Comparative Example 4). The blood collection volume of Example, Comparative Example 1, and Comparative Example 4 was 10 mL. Due to the limitations of commercially available product specifications, the blood collection volumes of Comparative Example 2 and Comparative Example 3 were 5 mL and 5.5 mL, respectively. To ensure comparability between groups, a paired design with uniform centrifugation / sampling rules was used, and the evaluation index was "plasma recovery rate = plasma volume / whole blood volume × 100%". This index is not affected by the absolute blood volume and can objectively reflect the plasma recovery capacity of different casts.

[0037] The experimental steps are as follows: After blood collection from all blood collection tubes, centrifugation was performed once under the following conditions: Examples 1, 2, and 4 were centrifuged at 1600×g for 10 min, while Comparative Example 3 was centrifuged at its publicly recommended 1800×g for 10 min. After centrifugation, plasma samples were obtained according to the standard operating procedures for each tube type: Comparative Examples 1 and 4 required a second centrifugation (16000×g, 10 min) to remove residual cells and debris, and then the supernatant plasma was aspirated; Comparative Example 2 directly aspirated the supernatant plasma after centrifugation; Comparative Example 3 and Examples, having already formed mechanical separation or isolation structures after centrifugation, did not require a second centrifugation, and the supernatant plasma was directly aspirated. The plasma volume obtained in each group was measured, and the plasma recovery ratio was calculated. The results are as follows: Figure 4 As shown.

[0038] from Figure 4As can be seen, the plasma recovery rate of the embodiment is the highest, reaching approximately 55%; the plasma recovery rate of Comparative Example 3 is approximately 51%; the plasma recovery rate of Comparative Example 2 is approximately 49%; the plasma recovery rate of Comparative Example 4 is approximately 45%; and the plasma recovery rate of Comparative Example 1 is the lowest, at only 41%. The plasma recovery rate of the embodiment is significantly higher than that of the other comparative examples, which is mainly due to the structural design of the present invention: through the positioning function of the third tube section 113, the three-state switching function of the valve body 12, and the precise matching of the volume of the separating gel 23 and the second tube section 112, it is ensured that the plasma 21 after centrifugation is mainly concentrated in the first tube section 111, avoiding the plasma 21 from being locked and wasted after entering the second tube section 112; at the same time, the mechanical isolation of the valve body 12 reduces the risk of interfacial backmixing and cellular component upwelling, so that there is no need to sacrifice plasma volume to avoid the white film layer or deposited cells during sampling, thereby achieving a higher plasma recovery rate. Although Comparative Example 3 adopted a mechanical separation structure, its internal mechanical separator occupied a large area and lacked a precise matching design between the separating gel and the volume, resulting in a slightly lower plasma recovery rate than the example. Comparative Example 2 relied solely on the separating gel to form a barrier, which resulted in poor interface stability after centrifugation. During sampling, care had to be taken to avoid the separating gel and the white film layer, and some plasma could not be recovered due to its proximity to the interface, thus resulting in a low recovery rate. Comparative Examples 1 and 4 lacked an effective isolation structure after centrifugation. In order to obtain pure plasma samples, a second centrifugation was required or the interface had to be strictly avoided during sampling, resulting in the loss of some plasma and the lowest recovery rate.

[0039] The second step was a hemolysis degree comparison experiment to verify the ability of the device of this invention to inhibit hemolysis during transportation. This experiment used the hemoglobin absorption peak as a measure. Hemoglobin is produced by the rupture of red blood cells, and its value should reflect the degree of red blood cell rupture; it should be as low as possible. The experimental groups were the same as those in the plasma recovery ratio experiment, using a paired sampling method, with 10 samples per group. The experimental steps were as follows: After blood was collected from all blood collection tubes and centrifuged to separate layers, they were placed in a 25°C environment and subjected to simulated transportation for 48 hours using a shaking device. The shaking frequency was set to 100 times / minute to simulate the shaking environment in actual logistics. After the simulated transportation, plasma samples were obtained according to the standard procedure for each tube type. The absorbance of each group of plasma samples at 414 nm was measured using a Nanodrop One nucleic acid protein analyzer. This wavelength is the characteristic absorption peak of hemoglobin; higher absorbance indicates more severe hemolysis. The experimental results are as follows: Figure 5 As shown.

[0040] from Figure 5As can be seen, the plasma sample in the embodiment had the lowest absorbance at 414 nm, approximately 1.46; the absorbance of Comparative Example 3 was approximately 1.52; the absorbance of Comparative Example 4 was approximately 1.63; the absorbance of Comparative Example 2 was approximately 1.66; and the absorbance of Comparative Example 1 was the highest, approximately 1.82. The degree of hemolysis in the embodiment was significantly lower than that in the other comparative examples, which fully demonstrates that the device of the present invention has a good inhibitory effect on hemolysis. The reason for this is that the mechanical isolation formed by the valve body 12 at the third tube section 113, in conjunction with the interface barrier formed by the separating gel 23, achieves dual stable isolation between the plasma layer 21 and the blood cell layer 22. Under the oscillating conditions of simulated transportation, the mechanical impact on the red blood cells 221 is significantly reduced, and even if a small number of red blood cells 221 rupture, the hemoglobin released is difficult to cross the mechanical barrier and enter the plasma layer 21, thereby reducing the degree of hemolysis in the plasma sample. Comparative Example 3 employs a mechanical separation structure, which also has a certain inhibitory effect on hemolysis, but it lacks the synergistic barrier of the separating gel, and its degree of hemolysis is slightly higher than that of the Example. Comparative Example 2 relies solely on the separating gel to form a barrier. Under transport vibration conditions, the separating gel interface is easily disturbed, causing some red blood cell rupture products to enter the plasma, resulting in a relatively high degree of hemolysis. Comparative Example 1 lacks an effective isolation structure after centrifugation. The vibration during transport directly affects the interface between plasma and blood cells, resulting in the most severe mechanical damage to red blood cells and the highest degree of hemolysis. Although Comparative Example 4 contains a cell protection system, under long-term vibration conditions, the protective effect of its chemical stabilizer is limited, and a certain degree of hemolysis still exists.

[0041] The next step was a DNA fragment distribution comparison experiment to verify the advantages of the device of this invention in inhibiting gDNA contamination of cfDNA. The experimental groups were the same as those described above, and the simulated transport conditions were also consistent (25℃, 48h shaking). After transport, plasma samples from each group were obtained according to standard procedures, and cfDNA was extracted using the QIAamp Circulating Nucleic Acid Kit. Subsequently, the DNA fragment distribution was determined using the LabChip GX Touch nucleic acid analysis system. The peak in the approximately 170bp region was a characteristic peak of cfDNA, and the peaks or tails in the >500bp region served as indicative characteristics of large-molecule gDNA contamination. The experimental results are as follows: Figure 6 As shown.

[0042] from Figure 6It can be seen that all plasma samples showed obvious cfDNA characteristic peaks around 170bp, indicating that all tubing types could separate cfDNA. However, in the >500bp region, different tubing types showed significant differences: Example 1 showed no obvious peaks or tails in the >500bp region, indicating that the cfDNA in its plasma sample was almost uncontaminated by gDNA; Comparative Example 4 also showed no obvious peaks or tails in the >500bp region, which is because its chemical stabilizer inhibited cell lysis and reduced gDNA release; Comparative Example 3 showed a slight peak in the >500bp region, indicating that its mechanical separation structure had a certain inhibitory effect on gDNA contamination, but the effect was slightly less than that of Example 1; Comparative Example 2 showed a more obvious peak in the >500bp region, indicating that its separating gel barrier failed to completely prevent gDNA from surfacing under transport vibration conditions; Comparative Example 1 showed the most obvious peaks and tails in the >500bp region, indicating that the cfDNA in its plasma sample was severely contaminated by gDNA. This experimental result fully demonstrates that the device of this invention, through the synergistic effect of "mechanical isolation + separation gel barrier," can effectively inhibit the entry of gDNA released from cell rupture into the plasma during transportation, thereby ensuring the purity and integrity of cfDNA and providing a high-quality sample basis for subsequent gene detection and mutation analysis. While Comparative Example 4 also inhibits gDNA contamination, it relies on chemical stabilizers, which may potentially interfere with downstream detection. The present invention, however, does not require chemical stabilizers and achieves gDNA contamination inhibition through a purely physical method, making it more advantageous in application.

[0043] Finally, a methylation compatibility comparison experiment was conducted to verify whether the device of the present invention could maintain the stability of methylation detection results without introducing a chemical preservation system. Ten healthy volunteers were selected as the experimental subjects, and a paired design was adopted. Each volunteer used the separation device 1 of the present invention (example) and the Streck blood collection tube (comparative example 5) to collect two tubes of whole blood. The blood volume of each tube was 10 mL. One tube was used for "0h (immediately after collection) processing" and the other tube was used for "25°C, 48h simulated post-transport processing".

[0044] The experimental steps are as follows: For the 0h treatment group, immediately after blood collection, the two types of tubing were centrifuged (1600×g, 10min), and the supernatant plasma was collected. To ensure consistent clarity of plasma samples obtained from different tubing types and reduce the potential impact of residual cells or debris on methylated reads, all plasma samples were uniformly clarified and centrifuged (16000×g, 10min), and the supernatant plasma was collected for later use. For the 48h simulated transport post-treatment group, the blood collection tubes were placed at 25℃ and simulated transport was performed for 48 hours using a shaking device (shaking frequency 100 times / min). After transport, plasma samples were prepared according to the same procedure as the 0h treatment group. cfDNA was extracted from plasma samples of each group using the QIAamp Circulating Nucleic Acid Kit. Targeted methylation sequencing was performed on the extracted cfDNA. To reduce batch effects, 0h and 48h samples from the same volunteer were used in the same batch for library construction and sequencing, and the same bioinformatics analysis procedure was used to obtain methylated results. β value.

[0045] To evaluate the baseline stability of methylation readout, the metric consistent_methylation (mean methylation level of conserved hypermethylated sites) is defined: a set of conserved sites exhibiting long-term stable hypermethylation in plasma cfDNA is selected. The selection criteria for this set are: the average methylation level in independent healthy samples. β A value ≥ 0.95 and a variance / coefficient of variation below a preset threshold are required to ensure that this set can serve as a "baseline / scale" for methylation measurement systems. (The last part, "any site," appears to be incomplete and unrelated to the preceding sentence.) β The value is defined as: β =M / (M+U), where M is the number of methylated reads and U is the number of unmethylated reads. For each volunteer, the consistent_methylation (mean methylation level of conserved hypermethylated sites) value was calculated for the 0h and 48h samples, and pairwise comparisons were performed. The results were summarized as follows: Figure 7 As shown.

[0046] from Figure 7As can be seen, the consistent methylation (average methylation level of conserved hypermethylated sites) values ​​of the examples at 0h and 48h are basically consistent, with an average value of approximately 96.09. This indicates that after simulated transport at 25℃ for 48h, the methylation readout baseline of plasma cfDNA obtained by the device of the present invention remains stable without significant shift. In contrast, the consistent methylation (average methylation level of conserved hypermethylated sites) value of Comparative Example 5 (Streck blood collection tube) was approximately 95.93 at 0h, which decreased to approximately 92.44 after 48h, a decrease of approximately 3.49 percentage points, indicating that its methylation readout baseline shifted overall towards hypomethylation. This experimental result fully demonstrates the significant advantages of the device of the present invention: through purely physical mechanical isolation, the stability of plasma samples during transport can be achieved without relying on a chemical preservation system, avoiding the risk of methylation shift that may be caused by chemical stabilizers, ensuring the consistency and reliability of methylation detection results, and is particularly suitable for clinical diagnostic scenarios with high requirements for methylation measurement accuracy, such as early tumor screening and prognostic assessment.

[0047] Based on the above experimental results, the separation device 1 of the exemplary embodiment has the following significant technical advantages: First, the plasma recovery rate is high and stable. Through the synergistic design of the third tube section 113, the valve body 12, and the separating gel 23, the plasma 21 is prevented from being locked into the second tube section 112, and the plasma recovery rate reaches more than 55%, which is significantly higher than that of traditional blood collection tubes. Second, the hemolysis inhibition effect is good. The dual protection of mechanical isolation and the separating gel barrier effectively reduces the risk of hemolysis under transport shock conditions. The absorbance at 414nm is less than 1.5, which is better than other comparative tube types. Third, it can effectively inhibit gDNA contamination. The mechanical barrier formed after centrifugation prevents gDNA released by cell rupture from entering the plasma 21 layer. The cfDNA purity is high, and there is no obvious gDNA signal in the >500bp region. Fourth, it has strong compatibility for methylation detection. No chemical stabilizers are required, avoiding methylation shift. The methylation baseline remains stable after transport. Fifth, it is easy to operate and has high safety. Separation and isolation can be completed in one centrifugation without the need for tube transfer and secondary centrifugation, reducing the risk of biological contamination and leakage, and is suitable for a variety of application scenarios.

[0048] The various embodiments of exemplary models have been described above. These descriptions are exemplary and not exhaustive, and are not limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or improvement of the technology in the market, or to enable others skilled in the art to understand the various implementations disclosed herein.

[0049] The separation device 1 of the exemplary embodiment, through its ingenious structural design, solves many pain points in traditional blood separation technology, such as low plasma recovery rate, high risk of hemolysis, serious gDNA contamination, interference from chemical reagents with downstream detection, and cumbersome operation. It provides an efficient, reliable, and convenient blood separation solution for clinical diagnosis, biomedical research, and other fields. Its core innovation lies in integrating the "separation" and "isolation" functions into the same tube, which can be automatically completed with a single low-speed centrifugation, requiring no additional operation. This ensures sample quality while improving work efficiency, and has broad clinical application prospects and market value. In practical applications, the types and dosages of anticoagulants and nuclease inhibitors, as well as the density and volume of the separating gel 23, can be flexibly adjusted according to different detection needs, further expanding its application scope and providing strong support for the development of precision medicine. At the same time, the structural design of this invention also has good scalability. For example, the structure of the third tube section 113, the shape of the valve body 12, and the type of separating gel 23 can all be adjusted according to actual production and application needs. As long as the core functions of "pre-centrifugation flow, centrifugation stratification, and post-centrifugation isolation" can be achieved, they all fall within the protection scope of the exemplary embodiment.

[0050] The blood separation method and separation device 1 of the exemplary embodiment complement each other, featuring a simple process and convenient operation that can be completed without the need for specialized technicians. This reduces the requirements for the operating environment and personnel, facilitating its widespread application in primary healthcare institutions and on-site rapid diagnosis. This method is not only applicable to cfDNA detection but can also be widely used in various plasma-related testing fields such as proteomics analysis, hormone detection, and pathogen detection, demonstrating strong versatility. Furthermore, the use of the separation device 1 in cfDNA sample preparation provides a high-quality sample preparation tool for fields such as tumor liquid biopsy and non-invasive prenatal diagnosis, helping to improve diagnostic accuracy and sensitivity and promoting the advancement of precision medicine technology.

[0051] In summary, the separation device, blood separation method, and application of the exemplary embodiments, through innovative structural design and collaborative working mechanism, achieve high efficiency, automation, and high purity in blood separation, solving many defects of traditional technologies, providing important technical support for the development of related fields, and demonstrating significant technological progress and practical value.

[0052] The various embodiments have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical applications, or improvements to the technology in the market, or to enable others skilled in the art to understand the various implementations disclosed herein.

Claims

1. A separation device (1), comprising: Pipe body (11), including: The first pipe section (111) includes the pipe opening (115); and The second pipe segment (112) includes a bottom pipe (116) and communicates with the first pipe segment (111) via a third pipe segment (113), wherein the radial dimension of the third pipe segment (113) is smaller than that of the first pipe segment (111) and the second pipe segment (112); and Valve body (12) includes: The valve cover (121) has a maximum radial dimension smaller than that of the first pipe section (111) and a maximum radial dimension larger than that of the third pipe section (113), so that the valve cover (121) can move within the first pipe section (111) in a first direction (D1) from the pipe opening (115) to the pipe bottom (116) and be positioned at the third pipe section (113) under centrifugal force; A spacer (122) is provided on the bottom surface of the valve cover (121) on the side facing the second pipe section (112) and protrudes along the first direction (D1). The end of the spacer (122) away from the valve cover (122) abuts against the third pipe section (113); and An elastomer (123) is attached to the side of the valve cover (121) near the second pipe section (112).

2. The separation device (1) according to claim 1, wherein the valve body (12) is configured to: When the valve body (12) is in a non-centrifugal state, the elastic body (123) is in the first state; When the valve body (12) is in a centrifugal state, the elastic body (123) deforms under centrifugal force and extends into the third pipe section (113) and the second pipe section (112); and When the valve body (12) is in a centrifugal state, the elastic body (123) forms a compression fit and / or interference fit with the inner wall of the third pipe section (113) under the elastic recovery action, so that the valve body (12) is locked at the third pipe section (113) and the fluid communication between the first pipe section (111) and the second pipe section (112) is blocked.

3. The separation device (1) according to claim 1 or 2, wherein the third pipe section (113) comprises: The first reduction portion (1131) has its radial dimension reduced along the first direction (D1) to form the narrowest portion (1133); and The second reduction section (1132) has a radial dimension that is reduced along a second direction (D2) from the second pipe section (112) to the narrowest part (1133), the second direction (D2) being opposite to the first direction (D1).

4. The separation device (1) according to any one of claims 1 to 2, wherein the longitudinal dimension of the valve body (12) along the first direction (D1) is greater than the radial dimension of the first pipe section (111) and the second pipe section (112).

5. The separation device (1) according to any one of claims 1 to 2, wherein a separation gel (23) is pre-placed in the tube body (11), and the density of the separation gel (23) is between the plasma (21) of the blood (20) and the blood cells (22) of the blood (20), wherein the blood (20) enters the tube body (11) through the port (115).

6. The separation device (1) according to claim 5, wherein the volume of the separation gel (23) is set such that the sum of its volume and the volume of the blood cells entering the second tube segment (112) after centrifugation is not less than the volume of the second tube segment (112), so as to prevent the plasma from entering the second tube segment (112) and thus being locked.

7. The separation device (1) according to any one of claims 1 to 2, wherein the valve cover (121) comprises: The intermediate portion (1211) and the circumferential portion (1212) wherein the longitudinal dimension of the intermediate portion (1211) along the first direction (D1) is greater than the longitudinal dimension of the circumferential portion (1212) along the first direction (D1).

8. The separation device (1) according to any one of claims 1 to 2 further includes a tube cap (13) which is sealed at the tube opening (115).

9. A method for blood separation, comprising: Blood (20) is placed in the tube (11) of the separation device (1) according to any one of claims 1 to 8. Centrifugation is performed to position the valve body (12) in the third tube segment (113) under centrifugal action and lock it in the third tube segment (113) after centrifugation, thereby placing the plasma (21) of the blood (20) in the first tube segment (111) and mechanically isolating it from the blood cells (22) of the blood (20) located in the second tube segment (112); and The plasma (21) is obtained from the first tube segment (111).

10. Use of the separation device (1) according to any one of claims 1-8 in the preparation of samples of circulating free deoxyribonucleic acid (cfDNA).

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