Separation device for cell clusters in malignant pleural effusion and use method of separation device

Abstract

The invention discloses a device for separating cell clusters in malignant pleural effusion and a use method of the device, and belongs to the technical field of cell separation.Two clamping blocks get close to each other and are separated from a clamping groove, the locking state between a supporting base and a mounting base is relieved, then the supporting base and a plurality of centrifugal tubes can be taken out of a centrifugal machine, and when the centrifugal tubes are taken down, the centrifugal tubes are separated from the mounting base; as the central axis of the tube frame and the central axis of the supporting seat are in a parallel state, the centrifugal tubes are directly and vertically pulled out upwards, the multiple centrifugal tubes can be conveniently and synchronously disassembled, the conical seat moves downwards to extrude the two clamping blocks to be clamped into the clamping grooves, and the purpose of locking the supporting seat and the mounting seat is achieved; therefore, the purpose of rapidly assembling and disassembling the batch centrifugal tubes is achieved through the centrifugal machine, the supporting seats and the tube frames which are modularly designed, the two sets of supporting seats and the multiple tube frames are adopted for centrifugal work, the centrifugal tubes can be alternately assembled and disassembled, the time spent on assembling and disassembling the centrifugal tubes is shortened, and efficient and stable separation work on cell clusters is facilitated.

Description

Technical Field

[0001] The invention relates to the technical field of cell separation, in particular to a device for separating cell clusters in malignant pleural effusion and a method for using the device. Background Art

[0002] In the biomedical engineering industry, malignant pleural effusion is a common complication in patients with malignant tumors. Its pathological characteristics include accumulation of fluid in the pleural cavity, which usually contains tumor cells and related cell clusters. Separation and analysis of cell clusters in malignant pleural effusion not only helps to clarify the diagnosis of tumors, but also provides important basis for disease staging, evaluation of treatment effects and research on tumor invasion and metastasis mechanisms.

[0003] When dealing with malignant pleural effusion, the cell cluster separation device is an important tool that can help doctors and researchers separate tumor cells from pleural effusion for further diagnosis and treatment.

[0004] At present, the commonly used methods for separating cell clusters in malignant pleural effusion include but are not limited to centrifugation. Traditional centrifugation directly separates cells in the liquid through a single centrifugation step. However, since the density of cell clusters is similar to that of single cells and fragments, the separation efficiency is low and it is easy to cause the loss of cell clusters. Especially in the face of complex pleural effusion samples, it is difficult for existing methods to efficiently recover complete cell clusters. When using a centrifuge to separate cell clusters, centrifuge tubes containing samples need to be placed in the centrifuge one by one and tilted in the centrifuge tank, which is inconvenient to operate and increases the workload. In addition, the integrated design of the centrifuge and centrifuge tank makes it difficult to achieve the purpose of rapid loading and unloading of batches of centrifuge tubes. Each time the centrifuge tubes are loaded and unloaded, the centrifuge is in an inoperative state and takes a lot of time, which is not conducive to the efficient and stable separation of cell clusters. Summary of the invention

[0005] The object of the present invention is to provide a device for separating cell clusters in malignant pleural effusion and a method for using the device, so as to solve the problem that the existing method proposed in the above-mentioned background technology is difficult to efficiently recover complete cell clusters; when using a centrifuge to separate cell clusters, centrifuge tubes containing samples need to be placed in the centrifuge one by one and placed obliquely in the centrifuge tank, which is inconvenient to operate and increases the workload, and the centrifuge and centrifuge tank with an integrated design are difficult to achieve the purpose of fast loading and unloading of batches of centrifuge tubes, and the centrifuge is in an inoperative state each time the centrifuge tubes are loaded and unloaded, and a lot of time is consumed.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] A cell cluster separation device in malignant pleural effusion comprises a centrifuge, wherein a storage bin is provided on the centrifuge, a support base is provided in the storage bin, a mounting base is connected to the bottom of the support base, a centrifugal drive mechanism is installed at the bottom of the inner wall of the centrifuge, two hinges are installed on the back of the centrifuge, and a cover is installed on the centrifuge through the two hinges. A plurality of pipe racks are provided on the outer side of the support base, two limit assemblies are installed in the pipe rack, and a centrifugal tube is provided in the middle of the two limit assemblies. A support rod is hingedly connected to the lower part of the outer wall of the pipe rack through a pin shaft, and one end of the plurality of support rods close to each other passes through the support base and is hingedly connected to an adjustment assembly, the adjustment assembly is installed inside the support base, and sliding holes are respectively provided at positions corresponding to the support rods outside the support base, and the support rods pass through and slide in the sliding holes, an extrusion assembly is provided inside the adjustment assembly, and two locking assemblies are overlapped at the bottom end of the extrusion assembly, a square groove is provided at the bottom of the support base, and limit grooves are respectively provided on both sides of the square groove, and the locking assembly slides in the limit grooves.

[0008] As a further solution of the present invention, a groove and a circular groove are provided on the mounting seat, the bottom of the support seat is overlapped in the groove, the square groove at the bottom of the support seat is arranged outside the rectangular protrusion at the top of the mounting seat, and card grooves are respectively provided on both sides of the inner wall of the circular groove, and the locking assembly is card-connected in the card groove.

[0009] As a further solution of the present invention, the limit assembly includes a splint, which is arc-shaped and has a guide plate fixed on the top, a plurality of limit rods are fixed to the outer wall of the splint, the limit rods slide through the pipe rack, a first spring is connected to the outer sleeve of the limit rod, the two ends of the first spring are respectively fixed to the inner wall of the pipe rack and the outer wall of the splint, the centrifuge tube is located between two opposite splints, and a hinge seat is installed above the outer wall of the pipe rack, and the hinge seat is connected to the outer wall of the support seat.

[0010] As a further solution of the present invention, the adjustment assembly includes a stud, support bearings are respectively installed on both sides of the outer wall of the stud, the two support bearings are respectively clamped at the bottom and top of the inner wall of the support seat, the middle part of the stud is threadedly connected with a nut, a sleeve is fixed outside the nut, the end of the support rod is hinged to the outer wall of the sleeve through a pin shaft, and the top of the stud is fixedly connected with a turntable.

[0011] As a further solution of the present invention, a screw is threadedly connected to the edge of the turntable, the bottom end of the screw is overlapped on the top of the support seat, and a rotating sleeve is installed on the top of the screw.

[0012] As a further solution of the present invention, the extrusion assembly includes a screw rod, which is arranged through the inside of the stud, a turning handle is fixed to the top of the screw rod, a threaded sleeve is connected to the external thread of the screw rod, and the threaded sleeve is clamped at the bottom of the inner wall of the support seat, and a connecting bearing is provided at the bottom of the connecting bearing, and a conical seat is installed at the bottom of the connecting bearing, and the outer walls of the conical seat are respectively overlapped with the two locking assemblies.

[0013] As a further solution of the present invention, the locking assembly includes a card block, which is slidably connected in the limit groove. The card block is clamped in the card slot and the other side overlaps with the conical seat. A sliding rod slides through the card block, and the sliding rod is fixed on both sides of the inner wall of the limit groove. A second spring is connected to the outer shell of the sliding rod, and the second spring is fixed between the card block and the inner wall of the limit groove.

[0014] A method for using a device for separating cell clusters in malignant pleural effusion, the method comprising the following steps:

[0015] When taking out the centrifuge tube from the storage bin in the centrifuge, the cover is opened to expose the centrifuge tube and the support seat. The screw rod is driven to rotate by turning the handle on the turntable. The screw rod can move upward during the rotation of the threaded sleeve. The screw rod drives the conical seat upward through the connecting bearing. At the same time, the two clamping blocks slide on the outer wall of the conical seat. When the clamping block is separated from the conical seat, the clamping block is driven to slide in the limiting groove by the pulling force of the second spring, so that the two clamping blocks can approach each other and separate from the clamping groove of the inner wall of the circular groove, thereby releasing the locking state between the support seat and the mounting seat. The support seat can be lifted up to remove it from the mounting seat, and then the support seat and multiple centrifuge tubes can be taken out of the centrifuge. When taking out the centrifuge tube, because the central axis of the tube rack and the support seat is in a parallel state, the centrifuge tube is directly pulled out vertically upward, and then multiple centrifuge tubes can be removed synchronously.

[0016] When the centrifuge tube is installed inside the tube rack, the centrifuge tube is inserted from the top of the tube rack, and the bottom end of the centrifuge tube contacts the two guide pieces. Because the two guide pieces are combined into a Y-shaped design, the centrifuge tube can open the two guide pieces and the two clamping plates. After the centrifuge tube is inserted, the clamping plates are supported by the elastic force of the first spring, so that the clamping plates drive the limit rods to move in the tube rack, so that the two clamping plates are close to each other and clamp the centrifuge tube to limit the position. The flexible clamping method can limit the centrifuge tube and prevent the centrifuge tube from being damaged by excessive pressure.

[0017] When the support seat, the tube rack and the centrifuge tube are placed inside the centrifuge, the support seat is lifted to clamp the square groove at the bottom outside the rectangular protrusion on the top of the mounting seat, and the bottom of the support seat is limited by the groove to improve the installation stability. When the support seat and the mounting seat are locked, the handle is rotated in the opposite direction to drive the screw rod to rotate. During the rotation of the screw rod, the conical seat can be driven to move downward through the connecting bearing, and the conical seat can squeeze the two clamping blocks to move them away from each other until the clamping blocks are clamped in the clamping groove, thereby achieving the purpose of locking the support seat and the mounting seat;

[0018] Before centrifugation, the centrifuge tube is placed inside the centrifuge at a fixed angle, so that the centrifuge tube forms a fixed angle with the rotating axis during the centrifugation process, which helps the sample particles slide along the tube wall of the centrifuge tube to the bottom to form a sediment, making the sediment more compact. Therefore, by holding the rotating sleeve and rotating the turntable, the turntable drives the stud to rotate. The screw rod is located in the hole opened in the middle of the stud. Therefore, the screw rod will not be driven to rotate synchronously during the rotation of the stud. During the rotation of the stud, the nut can drive the ferrule to move downward. When the ferrule moves downward, it can push the support rod to deflect. The support rod pushes the tube rack to deflect around the hinge seat, and the support rod is limited by the sliding hole to improve the stability of the support rod driving the tube rack to deflect. Then, multiple tube racks will drive multiple centrifuge tubes to deflect synchronously, so that the centrifuge tube is placed inside the centrifuge at a fixed angle. Secondly, tighten the screw to make it move downward and contact with the top of the support seat, so as to achieve the purpose of locking the turntable and prevent the turntable from rotating during the centrifugation process.

[0019] When centrifuging the centrifuge tube containing the malignant pleural effusion sample, close the machine cover and control the centrifuge drive mechanism to work. The specific steps are as follows:

[0020] Step 1: Centrifuge the tube at 1500 r / min for 10 minutes, collect the sediment at the bottom of the centrifuge tube, pour out the supernatant, and keep the sediment for the next step;

[0021] Step 2: Add the precipitate to the corresponding volume of lysed red blood cell solution and allow to react at room temperature for 15 minutes. After lysis is complete, centrifuge at 1500r / min for 5 minutes to remove the lysed red blood cell fragments and other impurities, and collect the precipitate;

[0022] Step 3: Resuspend the pellet obtained in step 2 in 50 mL of PBS buffer and then perform gradient centrifugation according to the following centrifugation procedure:

[0023] First step: centrifugation: centrifuge at 800r / min for 5 minutes, collect the precipitate, resuspend it in 50mL PBS, and use it as the sample for the next step of centrifugation. After centrifugation, collect the supernatant separately for use in free cell separation;

[0024] Second step centrifugation: Centrifuge the sample resuspended in the first step at 600r / min for 5 minutes to obtain a new precipitate, and resuspend the precipitate in 50mL PBS again as the sample for the next step centrifugation;

[0025] The third step is centrifugation: the resuspended sample in the second step is centrifuged at 400 r / min for 5 minutes to obtain the bottom precipitate, and the precipitate is resuspended and centrifuged again at 400 r / min;

[0026] When taking out the centrifuge tube from the centrifuge after completing the centrifugal work, open the machine cover and loosen the screws so that the screws can be separated from the support seat, and the turntable is rotated in the opposite direction by the rotating sleeve, so that the turntable drives the lead screw to rotate, so that the nut can drive the ferrule to move upward, and the ferrule pulls several support rods to deflect synchronously, so that the support rods pull the bottom of the tube rack close to the support seat, until the tube rack drives the centrifuge tube and the central axis of the support seat to be parallel, and after the support seat is taken out of the centrifuge, the centrifuge tube can be pulled out vertically upward.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] 1. The present invention drives the screw to rotate by rotating the handle on the turntable. The screw can move upward during the rotation of the threaded sleeve. The screw drives the conical seat upward through the connecting bearing. At the same time, the two clamping blocks slide on the outer wall of the conical seat. In the process of the clamping block detaching from the conical seat, the clamping block is driven to slide in the limiting groove by the pulling force of the second spring, so that the two clamping blocks can approach each other and detach from the clamping groove on the inner wall of the circular groove, thereby releasing the locking state between the support seat and the mounting seat. The support seat can be lifted up to remove it from the mounting seat, and then the support seat and multiple centrifuge tubes can be taken out of the centrifuge. When removing the centrifuge tube, because the central axis of the tube rack and the support seat are in a parallel state, the centrifuge tube is directly pulled out vertically upward, so that the multiple centrifuge tubes can be removed synchronously, and the support seat, the tube rack and the centrifuge tube are placed When the centrifuge is inside, the support seat is lifted to clamp the square groove at the bottom onto the rectangular protrusion on the top of the mounting seat. The bottom of the support seat is limited by the groove to improve the stability of the installation. When the support seat and the mounting seat are locked, the handle is rotated in the opposite direction to drive the screw to rotate. During the rotation of the screw, the conical seat can be driven downward by the connecting bearing. The conical seat can squeeze the two blocks to move away from each other until the blocks are inserted into the grooves, thereby achieving the purpose of locking the support seat and the mounting seat. Therefore, the modularly designed centrifuge, support seat and tube rack can achieve the purpose of fast loading and unloading of batch centrifuge tubes. Two sets of support seats and multiple tube racks are used for centrifugation, so that the centrifuge tubes can be loaded and unloaded alternately, shortening the time spent on loading and unloading the centrifuge tubes, which is conducive to the efficient and stable separation of cell clusters.

[0029] 2. The present invention holds the rotating sleeve and rotates the turntable, so that the turntable drives the stud to rotate. The screw rod is located in the hole opened in the middle of the stud. Therefore, the screw rod will not be driven to rotate synchronously during the rotation of the stud. During the rotation of the stud, the nut can drive the ferrule to move downward. When the ferrule moves downward, it can push the support rod to deflect. The support rod pushes the tube rack to deflect around the hinge seat, and the support rod is limited by the sliding hole, which improves the stability of the support rod driving the tube rack to deflect. Then, multiple tube racks will drive multiple centrifuge tubes to deflect synchronously, so that the centrifuge tubes are placed inside the centrifuge at a fixed angle, which helps the sample particles to slide to the bottom along the tube wall of the centrifuge tube to form precipitation, making the precipitation more compact. By adjusting the angle of the tube rack, the centrifugal effect is improved, and the central axis of the centrifuge tube and the support seat can be parallel to each other, which is convenient for taking and placing multiple centrifuge tubes at the same time, thereby improving the convenience of operation.

[0030] 3. The present invention inserts the centrifuge tube from the top of the tube rack when installing the centrifuge tube inside the tube rack, and the bottom end of the centrifuge tube contacts the two guide plates. The two guide plates are combined into a Y-shaped design, so that the centrifuge tube can open the two guide plates and the two clamps. After the centrifuge tube is inserted, the clamp is supported by the elastic force of the first spring, so that the clamp drives the limit rod to move in the tube rack, so that the two clamps are close to each other and clamp the centrifuge tube to limit the position. While limiting the centrifuge tube by flexible clamping, it can prevent the centrifuge tube from being damaged by excessive pressure, thereby protecting the centrifuge tube.

[0031] 4. The present invention uses a centrifugation procedure to perform gradient centrifugation when centrifuging a centrifuge tube containing a malignant pleural effusion sample, gradually reduces the centrifugal force and combines the precipitation and resuspension method in each step, thereby effectively avoiding the destruction of the cell cluster by high centrifugal force, ensuring the integrity and high recovery rate of the cell cluster. At the same time, the standardized operating procedure ensures the repeatability and extensibility of the method. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for describing the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative work.

[0033] Figure 1 It is a three-dimensional structural schematic diagram of the present invention;

[0034] Figure 2 It is a structural schematic diagram of a partial cross section of the centrifuge of the present invention;

[0035] Figure 3 It is a structural schematic diagram of the connection between the support seat and the mounting seat of the present invention;

[0036] Figure 4 It is a structural schematic diagram of the cross section of the pipe rack of the present invention;

[0037] Figure 5 For the present invention Figure 4 The enlarged structural diagram at A in the middle;

[0038] Figure 6 It is a structural schematic diagram of the cross section of the mounting seat of the present invention;

[0039] Figure 7 It is a structural schematic diagram of the mounting seat of the present invention;

[0040] Figure 8 It is a structural schematic diagram of the cross section of the support seat of the present invention;

[0041] Fig. 9 For the present invention Figure 8 The enlarged structural diagram at B in the middle;

[0042] Fig.10 It is a schematic structural diagram of the extrusion assembly of the present invention.

[0043] In the accompanying drawings, the components represented by the reference numerals are listed as follows:

[0044] 1. Centrifuge; 2. Storage compartment; 3. Support seat; 4. Mounting seat; 5. Centrifugal drive mechanism; 6. Groove; 7. Round groove; 8. Slot; 9. Pipe rack; 10. Limiting assembly; 101. Clamp; 102. Guide plate; 103. Limiting rod; 104. First spring; 11. Support rod; 12. Adjusting assembly; 121. Stud; 122. Nut; 123. Card sleeve; 124. Turntable; 125. Screw Nail; 126, rotating sleeve; 13, sliding hole; 14, supporting bearing; 15, extrusion assembly; 151, screw rod; 152, turning handle; 153, threaded sleeve; 154, connecting bearing; 155, conical seat; 16, locking assembly; 161, block; 162, sliding rod; 163, second spring; 17, limiting groove; 18, square groove; 19, machine cover; 20, hinge; 21, centrifuge tube; 22, hinged seat. DETAILED DESCRIPTION

[0045] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0046] See also Figure 1-Figure 10 , the present invention provides a technical solution:

[0047] A device for separating cell clusters in malignant pleural effusion comprises a centrifuge 1. A storage bin 2 is provided on the centrifuge 1. A support seat 3 is provided in the storage bin 2. A mounting seat 4 is connected to the bottom of the support seat 3. A centrifugal drive mechanism 5 is installed at the bottom of the inner wall of the centrifuge 1. Two hinges 20 are installed on the back of the centrifuge 1. A cover 19 is installed on the centrifuge 1 through the two hinges 20.

[0048] A plurality of pipe racks 9 are provided on the outside of the support seat 3. A hinge seat 22 is installed above the outer wall of the pipe rack 9. The hinge seat 22 is rotatably connected to the outer wall of the top end of the support seat 3 through a pin. Two limit assemblies 10 are installed in the pipe rack 9. The limit assemblies 10 include a clamping plate 101. The clamping plate 101 is of arc design and a guide sheet 102 is fixed on the top. A plurality of limit rods 103 are fixed on the outer wall of the clamping plate 101. The limit rods 103 penetrate and slide in the pipe rack 9. The limit rods 103 are outer-mounted with a first spring 104. The two ends of the first spring 104 are respectively fixed to the inner wall of the pipe rack 9 and the outer wall of the clamping plate 101. The centrifuge tube 21 is located between the two opposite clamping plates 101.

[0049] Because the two guide plates 102 are combined into a Y-shaped design, the centrifuge tube 21 can open the two guide plates 102 and the two clamps 101. The clamps 101 are supported by the elastic force of the first spring 104, so that the clamps 101 drive the limiting rods 103 to move in the tube rack 9, so that the two clamps 101 are close to each other and clamp the centrifuge tube 21 to limit the position. The flexible clamping method can not only limit the centrifuge tube 21, but also prevent the centrifuge tube 21 from being damaged by excessive pressure.

[0050] A support rod 11 is hinged to the lower part of the outer wall of the pipe rack 9 through a pin shaft, and one end of a plurality of support rods 11 close to each other passes through the support seat 3 and is hinged to an adjustment assembly 12, which is installed inside the support seat 3. As a further solution of the present invention, the adjustment assembly 12 includes a stud 121, and support bearings 14 are respectively installed on both sides of the outer wall of the stud 121, and the two support bearings 14 are respectively clamped at the bottom and top of the inner wall of the support seat 3. A nut 122 is threadedly connected to the middle part of the stud 121, and a sleeve 123 is fixed outside the nut 122. The end of the support rod 11 is hinged to the outer wall of the sleeve 123 through a pin shaft, and a turntable 124 is fixed to the top of the stud 121. Slide holes 13 are respectively opened at the positions corresponding to the support rods 11 outside the support seat 3, and the support rods 11 pass through and slide in the slide holes 13.

[0051] During the rotation of the stud 121, the nut 122 can drive the sleeve 123 to move downward. When the sleeve 123 moves downward, it can push the support rod 11 to deflect. The support rod 11 pushes the pipe rack 9 to deflect around the hinge seat 22, and then the multiple pipe racks 9 will drive the multiple centrifuge tubes 21 to deflect synchronously, which is convenient for the synchronous adjustment of the angles of the multiple centrifuge tubes 21.

[0052] An extrusion assembly 15 is arranged inside the adjustment assembly 12, and the extrusion assembly 15 includes a screw rod 151. The screw rod 151 is located in a hole opened in the middle of the stud 121. The screw rod 151 is arranged inside the stud 121, so the screw rod 151 will not be driven to rotate synchronously during the rotation of the stud 121. A turning handle 152 is fixed to the top of the screw rod 151. The screw rod 151 is externally threaded with a threaded sleeve 153. The threaded sleeve 153 is clamped at the bottom of the inner wall of the support seat 3. A connecting bearing 154 is provided at the bottom of the screw rod 151, and a conical seat 155 is installed at the bottom of the connecting bearing 154.

[0053] The edge of the turntable 124 is threadedly connected with a screw 125, the bottom end of the screw 125 overlaps the top of the support seat 3, and a rotating sleeve 126 is installed on the top of the screw 125. By tightening the screw 125 to move it downward and contact the top of the support seat 3, the turntable 124 is locked to prevent the turntable 124 from rotating during the centrifugation process.

[0054] The bottom of the support seat 3 is provided with a square groove 18, and the two sides of the square groove 18 are respectively provided with limit grooves 17, and the locking assembly 16 slides in the limit groove 17. The mounting seat 4 is provided with a groove 6 and a circular groove 7, and the bottom of the support seat 3 is overlapped in the groove 6, and the square groove 18 at the bottom of the support seat 3 is arranged outside the rectangular protrusion at the top of the mounting seat 4. The inner wall of the circular groove 7 is provided with a clamping groove 8 on both sides, and the locking assembly 16 is clamped in the clamping groove 8.

[0055] By clamping the square groove 18 at the bottom of the support seat 3 onto the rectangular protrusion on the top of the mounting seat 4 and limiting the bottom of the support seat 3 through the groove 6, the installation stability is improved, and when the centrifugal drive mechanism 5 drives the mounting seat 4 to rotate, the stability of the rotation of the support seat 3 and the centrifuge tube 21 driven by the mounting seat 4 can be ensured.

[0056] As a further solution of the present invention, the outer walls of the conical seat 155 are overlapped with the two locking components 16 respectively, and the overlapping locking components 16 include a card block 161, which is slidably connected in the limit groove 17, and the card block 161 is clamped in the card groove 8 and the other side is overlapped with the conical seat 155, and a sliding rod 162 slides through the card block 161, and the sliding rod 162 is fixed on both sides of the inner wall of the limit groove 17. The sliding rod 162 is outer-connected with a second spring 163, and the second spring 163 is fixed between the card block 161 and the inner wall of the limit groove 17.

[0057] Since the screw rod 151 is threadedly connected in the threaded sleeve 153, and the threaded sleeve 153 is clamped at the bottom of the inner wall of the support seat 3, the screw rod 151 can move downward during the rotation process, and then the screw rod 151 drives the conical seat 155 to move downward through the connecting bearing 154 during the movement process, and the conical seat 155 can squeeze the two blocks 161 to make them move away from each other, so as to facilitate the synchronous adjustment of the positions of the two blocks 161. The block 161 is driven to slide on the limit groove 17 and the slide rod 162 by the pulling force of the second spring 163, so as to improve the stability of the horizontal movement of the block 161, so that the two blocks 161 can approach each other and break away from the groove 8 on the inner wall of the circular groove 7, so as to facilitate the release of the locking state between the support seat 3 and the mounting seat 4. When the screw rod 151 rotates inside the connecting bearing 154, the conical seat 155 and the block 161 generate friction, so as to make the conical seat 155 not rotate synchronously with the screw rod 151, so as to improve the stability of the adjustment of the block 161.

[0058] During operation, the purpose of fast loading and unloading of batch centrifuge tubes 21 is achieved through the modularly designed centrifuge 1, support base 3 and tube rack 9. Two sets of support bases 3 and multiple tube racks 9 are used to perform centrifugation work, so that the centrifuge tubes 21 can be alternately loaded and unloaded, shortening the time spent on loading and unloading the centrifuge tubes 21, which is beneficial to the efficient and stable separation of cell clusters.

[0059] A method for using a device for separating cell clusters in malignant pleural effusion, the method comprising the following steps:

[0060] When taking out the centrifuge tube 21 from the storage bin 2 in the centrifuge 1, the machine cover 19 is opened to expose the centrifuge tube 21 and the support seat 3 to the outside, and the screw rod 151 is driven to rotate by rotating the handle 152 on the turntable 124. The screw rod 151 can move upward during the rotation inside the threaded sleeve 153, and the screw rod 151 drives the conical seat 155 to move upward through the connecting bearing 154, and at the same time, the two blocks 161 slide on the outer wall of the conical seat 155. In the process of the clamping block 161 being separated from the conical seat 155, the clamping block 161 is driven to slide in the limiting groove 17 by the pulling force of the second spring 163, so that the two clamping blocks 161 can approach each other and separate from the clamping groove 8 on the inner wall of the circular groove 7, thereby releasing the locking state between the support seat 3 and the mounting seat 4, and lifting the support seat 3 to remove it from the mounting seat 4, and then the support seat 3 and the multiple centrifuge tubes 21 can be taken out from the centrifuge 1. When removing the centrifuge tube 21, because the central axis of the tube rack 9 and the support seat 3 is in a parallel state, the centrifuge tube 21 is directly pulled out vertically upward, and then the multiple centrifuge tubes 21 can be removed simultaneously;

[0061] When the centrifuge tube 21 is installed inside the tube rack 9, the centrifuge tube 21 is inserted from the top of the tube rack 9, and the bottom end of the centrifuge tube 21 contacts the two guide pieces 102. The two guide pieces 102 are combined into a Y-shaped design, so that the centrifuge tube 21 can open the two guide pieces 102 and the two clamps 101. After the centrifuge tube 21 is inserted, the clamps 101 are supported by the elastic force of the first spring 104, so that the clamps 101 drive the limiting rods 103 to move in the tube rack 9, so that the two clamps 101 are close to each other and clamp the centrifuge tube 21 to limit the position. While limiting the centrifuge tube 21 by flexible clamping, it can prevent the centrifuge tube 21 from being damaged by excessive pressure.

[0062] When the support seat 3, the tube rack 9 and the centrifugal tube 21 are placed inside the centrifuge 1, the support seat 3 is lifted to clamp the square groove 18 at the bottom onto the rectangular protrusion on the top of the mounting seat 4. The bottom of the support seat 3 is limited by the groove 6 to improve the installation stability. When the support seat 3 and the mounting seat 4 are locked, the handle 152 is rotated in the opposite direction to drive the screw rod 151 to rotate. During the rotation of the screw rod 151, the conical seat 155 can be driven downward by the connecting bearing 154. The conical seat 155 can squeeze the two blocks 161 to move away from each other until the blocks 161 are inserted into the slot 8, thereby achieving the purpose of locking the support seat 3 and the mounting seat 4.

[0063] Before centrifugation, the centrifuge tube 21 is placed inside the centrifuge 1 at a fixed angle, so that the centrifuge tube 21 forms a fixed angle with the rotation axis during the centrifugation process, which helps the sample particles slide along the tube wall of the centrifuge tube 21 to the bottom to form a sediment, making the sediment more compact. Therefore, by holding the rotating sleeve 126 and rotating the turntable 124, the turntable 124 drives the stud 121 to rotate. The screw rod 151 is located in the hole opened in the middle of the stud 121. Therefore, the screw rod 151 will not be driven to rotate synchronously during the rotation of the stud 121. During the rotation of the stud 121, the nut 122 can drive The ferrule 123 moves downward, and when the ferrule 123 moves downward, it can push the support rod 11 to deflect, and the support rod 11 pushes the pipe rack 9 to deflect around the hinge seat 22, and the support rod 11 is limited by the sliding hole 13, so as to improve the stability of the support rod 11 driving the pipe rack 9 to deflect, and then multiple pipe racks 9 will drive multiple centrifuge tubes 21 to deflect synchronously, so that the centrifuge tubes 21 are placed inside the centrifuge 1 at a fixed angle, and then the screw 125 is tightened to make it move downward and contact with the top of the support seat 3, so as to achieve the purpose of locking the turntable 124, and prevent the turntable 124 from rotating during the centrifugation process;

[0064] When the centrifuge tube 21 containing the malignant pleural effusion sample is centrifuged, the machine cover 19 is closed and the centrifuge drive mechanism 5 is controlled to work. The specific steps are as follows:

[0065] Step 1: The centrifugal drive mechanism 5 is centrifuged at 1500 r / min for 10 minutes, the sediment at the bottom of the centrifuge tube 21 is collected, the supernatant is poured out, and the sediment is retained for the next step;

[0066] Step 2: Add the precipitate to the corresponding volume of lysed red blood cell solution and allow to react at room temperature for 15 minutes. After lysis is complete, centrifuge at 1500r / min for 5 minutes to remove the lysed red blood cell fragments and other impurities, and collect the precipitate;

[0067] Step 3: Resuspend the pellet obtained in step 2 in 50 mL of PBS buffer and then perform gradient centrifugation according to the following centrifugation procedure:

[0068] First step: centrifugation: centrifuge at 800r / min for 5 minutes, collect the precipitate, resuspend it in 50mL PBS, and use it as the sample for the next step of centrifugation. After centrifugation, collect the supernatant separately for use in free cell separation;

[0069] Second step centrifugation: Centrifuge the sample resuspended in the first step at 600r / min for 5 minutes to obtain a new precipitate, and resuspend the precipitate in 50mL PBS again as the sample for the next step centrifugation;

[0070] The third step is centrifugation: the resuspended sample in the second step is centrifuged at 400 r / min for 5 minutes to obtain the bottom precipitate, and the precipitate is resuspended and centrifuged again at 400 r / min;

[0071] When the centrifuge tube 21 is taken out from the centrifuge 1 after the centrifugal work is completed, the machine cover 19 is opened and the screw 125 is loosened so that the screw 125 can be separated from the support seat 3, and the turntable 124 is rotated in the opposite direction by the rotating sleeve 126, so that the turntable 124 drives the screw rod 151 to rotate, so that the nut 122 can drive the ferrule 123 to move upward, and the ferrule 123 pulls a plurality of support rods 11 to deflect synchronously, so that the support rod 11 pulls the bottom of the pipe rack 9 close to the support seat 3, until the pipe rack 9 drives the centrifuge tube 21 to be parallel to the central axis of the support seat 3, and the centrifuge tube 21 can be vertically pulled out upward after the support seat 3 is taken out from the centrifuge 1.

Claims

1. A device for separating cell clusters in malignant pleural effusion, comprising a centrifuge (1), characterized in that: The centrifuge (1) is provided with a storage bin (2), a support seat (3) is provided in the storage bin (2), the bottom of the support seat (3) is connected to a mounting seat (4), a centrifugal drive mechanism (5) is installed at the bottom of the mounting seat (4), the centrifugal drive mechanism (5) is installed at the bottom of the inner wall of the centrifuge (1), two hinges (20) are installed on the back of the centrifuge (1), the centrifuge (1) is installed with a cover (19) through the two hinges (20), a plurality of pipe racks (9) are provided on the outer side of the support seat (3), two limit assemblies (10) are installed in the pipe rack (9), a centrifugal tube (21) is provided in the middle of the two limit assemblies (10), and the lower part of the outer wall of the pipe rack (9) is hinged by a pin. A support rod (11) is provided, and one end of a plurality of support rods (11) close to each other passes through a support seat (3) and is hingedly connected with an adjustment component (12); the adjustment component (12) is installed inside the support seat (3); sliding holes (13) are respectively provided at positions outside the support seat (3) corresponding to the support rods (11); the support rods (11) pass through and slide in the sliding holes (13); an extrusion component (15) is provided inside the adjustment component (12); two locking components (16) are overlapped at the bottom end of the extrusion component (15); a square groove (18) is provided at the bottom of the support seat (3); limiting grooves (17) are respectively provided on both sides of the square groove (18); the locking component (16) slides in the limiting grooves (17).

2. The device for separating cell clusters in malignant pleural effusion according to claim 1, characterized in that: The mounting seat (4) is provided with a groove (6) and a circular groove (7); the bottom of the support seat (3) is overlapped in the groove (6); the square groove (18) at the bottom of the support seat (3) is arranged outside the rectangular protrusion at the top of the mounting seat (4); the inner walls of the circular groove (7) are respectively provided with card slots (8); the locking assembly (16) is card-engaged in the card slots (8).

3. The device for separating cell clusters in malignant pleural effusion according to claim 1, characterized in that: The limiting assembly (10) comprises a clamping plate (101), the clamping plate (101) is of arc-shaped design and has a guide plate (102) fixed on the top, a plurality of limiting rods (103) are fixed on the outer wall of the clamping plate (101), the limiting rods (103) penetrate and slide in the pipe rack (9), a first spring (104) is connected to the outer surface of the limiting rod (103), the two ends of the first spring (104) are respectively fixed to the inner wall of the pipe rack (9) and the outer wall of the clamping plate (101), the centrifuge tube (21) is located between two opposite clamping plates (101), a hinge seat (22) is installed above the outer wall of the pipe rack (9), and the hinge seat (22) is connected to the outer wall of the support seat (3).

4. The device for separating cell clusters in malignant pleural effusion according to claim 2, characterized in that: The adjustment component (12) comprises a stud (121), support bearings (14) are respectively installed on both sides of the outer wall of the stud (121), and the two support bearings (14) are respectively clamped on the bottom and top of the inner wall of the support seat (3), a nut (122) is threadedly connected to the middle part of the stud (121), and a sleeve (123) is fixed outside the nut (122), the end of the support rod (11) is hinged to the outer wall of the sleeve (123) through a pin shaft, and the top of the stud (121) is fixedly connected to a turntable (124).

5. The device for separating cell clusters in malignant pleural effusion according to claim 4, characterized in that: A screw (125) is threadedly connected to the edge of the rotating disk (124), the bottom end of the screw (125) is overlapped on the top of the supporting seat (3), and a rotating sleeve (126) is installed on the top of the screw (125).

6. The device for separating cell clusters in malignant pleural effusion according to claim 4, characterized in that: The extrusion assembly (15) comprises a screw rod (151), the screw rod (151) is arranged to penetrate inside the stud (121), a rotating handle (152) is fixed to the top end of the screw rod (151), a threaded sleeve (153) is connected to the external thread of the screw rod (151), the threaded sleeve (153) is clamped to the bottom of the inner wall of the support seat (3), a connecting bearing (154) is provided at the bottom end of the screw rod (151), a conical seat (155) is installed at the bottom of the connecting bearing (154), and the outer wall of the conical seat (155) is overlapped with the two locking assemblies (16) respectively.

7. The device for separating cell clusters in malignant pleural effusion according to claim 6, characterized in that: The locking assembly (16) comprises a clamping block (161), wherein the clamping block (161) is slidably connected in the limiting groove (17), the clamping block (161) is clamped in the clamping groove (8) and the other side overlaps with the conical seat (155), a sliding rod (162) slides through the clamping block (161), the sliding rod (162) is fixed on both sides of the inner wall of the limiting groove (17), and a second spring (163) is connected to the outer shell of the sliding rod (162), and the second spring (163) is fixed between the clamping block (161) and the inner wall of the limiting groove (17).

8. A method for using a device for separating cell clusters in malignant pleural effusion, according to any one of claims 1 to 7, characterized in that: The method of use comprises the following steps: When taking out the centrifuge tube (21) from the storage bin (2) in the centrifuge (1), the cover (19) is opened to expose the centrifuge tube (21) and the support seat (3) to the outside, and the screw rod (151) is driven to rotate by rotating the handle (152) on the turntable (124). The screw rod (151) can move upward during the rotation inside the threaded sleeve (153). The screw rod (151) drives the conical seat (155) to move upward through the connecting bearing (154). At the same time, the two clamping blocks (161) slide on the outer wall of the conical seat (155). When the clamping block (161) is separated from the conical seat (155), the second spring (163) is used to release the clamping block (161). The pulling force drives the clamping block (161) to slide in the limiting groove (17), so that the two clamping blocks (161) can approach each other and disengage from the clamping groove (8) on the inner wall of the circular groove (7), thereby releasing the locking state between the support seat (3) and the mounting seat (4). The support seat (3) can be lifted up to remove it from the mounting seat (4), and then the support seat (3) and the plurality of centrifuge tubes (21) can be taken out from the centrifuge (1). When removing the centrifuge tube (21), because the central axis of the tube rack (9) and the support seat (3) are in a parallel state, the centrifuge tube (21) is directly pulled out vertically upward, and then the plurality of centrifuge tubes (21) can be removed simultaneously. When the centrifuge tube (21) is installed inside the tube rack (9), the centrifuge tube (21) is inserted from the top of the tube rack (9), and the bottom end of the centrifuge tube (21) contacts the two guide plates (102). Since the two guide plates (102) are combined into a Y-shaped design, the centrifuge tube (21) can open the two guide plates (102) and the two clamping plates (101). After the centrifuge tube (21) is inserted, the clamping plates (101) are supported by the elastic force of the first spring (104), so that the clamping plates (101) drive the limiting rod (103) to move inside the tube rack (9), so that the two clamping plates (101) are close to each other and clamp the centrifuge tube (21) to limit the position. While limiting the centrifuge tube (21) by a flexible clamping method, it can prevent the centrifuge tube (21) from being damaged due to excessive pressure. When the support seat (3), the tube rack (9) and the centrifuge tube (21) are placed inside the centrifuge (1), the support seat (3) is lifted to clamp the square groove (18) at the bottom onto the outside of the rectangular protrusion at the top of the mounting seat (4). The bottom of the support seat (3) is limited by the groove (6) to improve the installation stability. When the support seat (3) and the mounting seat (4) are locked, the handle (152) is rotated in the opposite direction to drive the screw rod (151) to rotate. During the rotation of the screw rod (151), the conical seat (155) can be driven to move downward through the connecting bearing (154). The conical seat (155) can squeeze the two clamping blocks (161) to move them away from each other until the clamping blocks (161) are clamped into the clamping groove (8), thereby achieving the purpose of locking the support seat (3) and the mounting seat (4). Before centrifugation, the centrifuge tube (21) is placed inside the centrifuge (1) at a fixed angle, so that the centrifuge tube (21) forms a fixed angle with the rotation axis during the centrifugation process, which helps the sample particles slide along the tube wall of the centrifuge tube (21) to the bottom to form a sediment, making the sediment more compact. Therefore, by holding the rotating sleeve (126) and rotating the turntable (124), the turntable (124) drives the stud (121) to rotate. The screw rod (151) is located in a hole opened in the middle of the stud (121). Therefore, the screw rod (151) will not be driven to rotate synchronously during the rotation of the stud (121). During the rotation of the stud (121), the nut (122) can drive the ferrule (121) to rotate. 23) moves downward, and when the ferrule (123) moves downward, it can push the support rod (11) to deflect, and the support rod (11) pushes the pipe rack (9) to deflect around the hinge seat (22), and the support rod (11) is limited by the sliding hole (13), thereby improving the stability of the support rod (11) driving the pipe rack (9) to deflect, and then multiple pipe racks (9) will drive multiple centrifuge tubes (21) to deflect synchronously, so that the centrifuge tubes (21) are placed inside the centrifuge (1) at a fixed angle, and then tighten the screw (125) to make it move downward and contact with the top of the support seat (3), so as to achieve the purpose of locking the turntable (124) and prevent the turntable (124) from rotating during the centrifugation process; When the centrifuge tube (21) containing the malignant pleural effusion sample is centrifuged, the machine cover (19) is closed and the centrifuge drive mechanism (5) is controlled to operate. The specific steps are as follows: Step 1: The centrifugal drive mechanism (5) is centrifuged at 1500 r / min for 10 minutes, the sediment at the bottom of the centrifuge tube (21) is collected, the supernatant is poured out, and the sediment is retained for the next step; Step 2: Add the precipitate to the corresponding volume of lysed red blood cell solution and allow to react at room temperature for 15 minutes. After lysis is complete, centrifuge at 1500r / min for 5 minutes to remove the lysed red blood cell fragments and other impurities, and collect the precipitate; Step 3: Resuspend the pellet obtained in step 2 in 50 mL of PBS buffer and then perform gradient centrifugation according to the following centrifugation procedure: First step: centrifugation: centrifuge at 800r / min for 5 minutes, collect the precipitate, resuspend it in 50mL PBS, and use it as the sample for the next step of centrifugation. After centrifugation, collect the supernatant separately for use in free cell separation; Second step centrifugation: Centrifuge the sample resuspended in the first step at 600r / min for 5 minutes to obtain a new precipitate, and resuspend the precipitate in 50mL PBS again as the sample for the next step centrifugation; The third step is centrifugation: the resuspended sample in the second step is centrifuged at 400 r / min for 5 minutes to obtain the bottom precipitate, and the precipitate is resuspended and centrifuged again at 400 r / min; When the centrifuge tube (21) is taken out of the centrifuge (1) after the centrifugation work is completed, the machine cover (19) is opened and the screw (125) is loosened so that the screw (125) can be separated from the support seat (3). The rotating disk (124) is rotated in the opposite direction by the rotating sleeve (126), so that the rotating disk (124) drives the screw rod (151) to rotate, so that the nut (122) can drive the clamping sleeve (123) to move upward, and the clamping sleeve (123) pulls a plurality of support rods (11) to deflect synchronously, so that the support rods (11) pull the bottom of the tube rack (9) close to the support seat (3), until the tube rack (9) drives the centrifuge tube (21) and the central axis of the support seat (3) to be in a parallel state, and after the support seat (3) is taken out of the centrifuge (1), the centrifuge tube (21) can be vertically drawn out upward.

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