A cell filter

Abstract

A cell filter, comprising a blood suction device, a blood filtration device and a blood recovery device. The blood recovery device includes a waste liquid bottle and a vacuum pump, and the vacuum pump is communicated with the waste liquid bottle. A blood suction needle is arranged inside the blood suction device. The blood filtration device is correspondingly provided with an upper sealing cavity, a movable support plate and a lower sealing cavity from bottom to top in sequence, and the lower sealing cavity is fixed on a lifting system. Blood passes through the blood suction needle of the blood suction device to the blood inflow needle of the blood filtration device, and then passes through a filter membrane and a blood recovery pipeline to the waste liquid bottle. The caliber of the blood suction needle is smaller than that of the blood inflow needle. In the present invention, a blood suction needle is arranged at the input end, a blood inflow needle is arranged at the output end, and the caliber of the suction needle is smaller than that of the inflow needle, so that blood can be filtered with extremely low negative pressure. The low pressure has little destructive effect on cells. A novel membrane pore structure is adopted, and the size and arrangement form of the pores are consistent, avoiding the pollution of the membrane pores by white blood cells.

Description

Technical Field

[0001] The present invention relates to the technical field of medical machinery, and particularly relates to a cell filter. Background Art

[0002] In the tumor cell capture technology based on cell morphology and size (ISET), the excessive negative pressure generated by the vacuum system is likely to cause the destruction and death of tumor cells. Currently, the detection of IEST requires pre-treatment of blood, using formaldehyde to fix cells, making the cells lose their vitality and unable to be used for the next-stage genomics research. The present invention does not require prior blood treatment, protects cells as much as possible, and at the same time the captured cells are live tumor cells.

[0003] CTC refers to tumor cells derived from tumor tissues and circulating in the blood. CTC is the target of chemotherapy and also the culprit of tumor metastasis and recurrence. Current research has confirmed that tumors release CTC into the patient's blood at the in-situ cancer stage, but most of them are cleared by the body's immune system. It is not until the tumor reaches a certain size that CTC can be detected in the patient's blood, but at this time, imaging still cannot detect the tumor. Therefore, monitoring CTC in the blood of high-risk populations has the opportunity to detect early tumors. However, at this time, the number of CTC in the blood is small, and a highly sensitive detection device is needed. The genomics detection of CTC can provide real-time tumor load information including gene mutations of patients, and can also be used for FISH chromosome recombination detection, NGS gene sequencing, PCR gene expression level detection, cell culture, etc., providing solutions for the precise treatment of tumor patients.

[0004] The capture and enrichment of CTC are based on principles and are divided into physical methods and chemical methods in total. The physical method is based on the morphology, size and density of CTC. The chemical method is based on specific markers of epithelial cells on the surface of CTC cells, and uses the specific reaction of the corresponding antibody and antigen wrapped by magnetic beads to achieve the purpose of separating CTC. According to the principle, it is divided into two methods: positive and negative. Due to more steps, the chemical method causes greater damage to CTC and is prone to cell apoptosis. The capture rate and purity of CTC are relatively low. is the only device currently approved by the US FDA for CTC separation. It separates CTC by the interaction between the antibody connected by magnetic beads and the marker antigen of epithelial cells on the surface of CTC. The disadvantage of this technology is that it is easy to cause CTC apoptosis, the number of detected CTC is small, the sensitivity is low, and it is easy to miss detecting EMT cells and CTC with negative expression of epithelial cell specific antigen. Exactly because of this, It has not become the standard instrument in the industry. Compared with the physical method based on cell morphology and size of the chemical method, the method is simple. Among the existing CTC capture and enrichment devices in China, the membrane-based products are the first-generation polycarbonate etched membranes. The pore size arrangement of this kind of membrane is disordered and the pore sizes are inconsistent, which easily leads to the accumulation of white blood cells on the membrane and pollutes the membrane, affecting the capture of CTC. Although this technology does not cause the apoptosis of CTC, the capture efficiency and purity of CTC are low, and the sensitivity is also low.

[0005] At present, most domestic gene testing companies focus on the targeted drug treatment of tumors, and there are relatively few involved in the early diagnosis or screening of tumors. The main reason is that the content of early CTC in the blood of tumors is very small, and there is a lack of highly sensitive detection methods. It takes about 5 to 10 years for a tumor to be undetectable to being detectable by imaging. When the volume of the tumor reaches 0.5 cm, it can be detected by imaging. At this time, the corresponding number of tumor cells is about 10 9 , that is, 1 billion. In fact, during the growth of a tumor, tumor cells are continuously released into the peripheral blood. Of course, in the early stage of the tumor, most of the tumor cells are cleared or shielded by the body's immune system. However, some tumor cells can still be detected in the blood. If CTC can be detected in the blood but no tumor is found by imaging examination; in this case, early tumors need to be detected by several consecutive tests.

[0006] The existing detection system enriches by applying membrane filtration according to the differences in cell size (ISET) and cell deformability between tumor cells and blood cells. There are nearly 200,000 round holes of the same size on the membrane, and the round holes are arranged in a certain specification, neatly and orderly. The membrane is placed at the bottom of a 6-ml cylindrical filter. The whole blood sample is added to the filter at one time. At this time, since the blood completely covers the membrane and all the holes on the membrane are blocked, a very large negative pressure is required. It is reported that a negative pressure of more than 400 kPa to 600 kPa (0.4 to 0.6 mpa) (industrial-grade vacuum pump) is required to allow the cells in the blood to pass through the filter membrane. In order to prevent the membrane from being completely deformed and the pore size from being distorted and blocked due to the adsorption of too high negative pressure, a layer of metal wire mesh is added under the membrane as a support pad for the membrane.

[0007] In summary, the existing related technologies have the following technical disadvantages:

[0008] 1) Excessive negative pressure filtration: In the ISET method, the whole blood sample is added to the filter at once, and all the pore diameters on the membrane are blocked. In this way, the negative pressure gradient in the cavity is eliminated, resulting in a zero pressure drop in the cavity. Therefore, a large external pressure (400 - 600 pka) is required to filter out the blood. Under such a large pressure, the pore diameter of the membrane deforms, and some tumor cells pass through the deformed pores and are filtered out, leading to a low capture efficiency of tumor cells. At the same time, accompanied by changes in cell structure, it will cause the death of some tumor cells.

[0009] 2) No living tumor cells: In order to reduce the damage of excessive negative pressure to blood cells and maintain the normal structure of cells, the blood must be pretreated. Generally, formaldehyde with different concentrations is used to fix the cells. In this way, there are no living tumor cells for the next-step genetic testing.

[0010] 3) In the ISET technology with a polycarbonate etched membrane, the size and arrangement of the membrane pores are inconsistent, which easily causes the accumulation of white blood cells on the membrane, contaminates the membrane, and results in a low CTC capture rate and capture purity.

[0011] 4) Low degree of automation: The blood filter is manually cleaned and cannot effectively handle large-scale sample detection. Summary of the Invention

[0012] A cell filter proposed by the present invention can solve the problem of how to capture living tumor cells in blood as much as possible under low pressure conditions.

[0013] To achieve the above object, the present invention adopts the following technical solutions:

[0014] A cell filter includes a housing, and further includes a blood suction device, a blood filtration device, and a blood recovery device respectively disposed in the housing. The blood recovery device includes a waste liquid bottle and a vacuum pump, and the vacuum pump is communicated with the waste liquid bottle;

[0015] The blood suction device is hermetically communicated with the inlet of the blood filtration device through a pipeline, and the outlet of the blood filtration device is hermetically communicated with the waste liquid bottle of the blood recovery device through a pipeline;

[0016] Among them,

[0017] A blood suction needle is arranged in the blood suction device;

[0018] The blood filtration device is correspondingly provided with an upper sealing cavity, a movable support plate, and a lower sealing cavity from top to bottom. The lower sealing cavity is fixed on the lifting system;

[0019] The upper sealing cavity includes an upper cavity body and an upper sealing ring fixed at the bottom of the upper cavity body. The top of the upper sealing cavity is hermetically connected with a blood inflow needle;

[0020] The movable support plate is placed between the upper sealing cavity and the lower sealing cavity. By starting the lifting system to move up and down, the lower sealing cavity and the movable support plate can be separated from and closed with the upper sealing cavity in sequence.

[0021] A filter hole is arranged at the center of the movable support plate, and a filter membrane is arranged on the filter hole.

[0022] The lower sealing cavity includes a lower cavity body and a lower sealing ring fixed on the top of the lower cavity body. The blood recovery pipeline inlet is hermetically communicated in the lower sealing cavity.

[0023] Blood is drawn from the blood suction needle of the blood suction device to the blood inflow needle of the blood filtration device, then drips onto the filter membrane and flows out, and flows into the waste liquid bottle through the blood recovery pipeline outlet.

[0024] The caliber of the blood suction needle is smaller than that of the blood inflow needle.

[0025] Further, the blood suction needle is fixed between the upper and lower rigid splints through a needle attachment device, and the upper and lower rigid splints are fixed on the housing.

[0026] Further, the needle attachment device is a hollow plastic structure with different sizes at both ends; the blood suction needle penetrates through the needle attachment device.

[0027] A spring is arranged in the upper half of the needle attachment device, and the needle is fixed between the two steel plates through the spring.

[0028] Further, an air outlet pipe is also arranged in the lower half of the needle attachment device.

[0029] Further, grooves are correspondingly arranged on the upper cavity body, the lower cavity body and the movable support plate for embedding sealing rings to maintain the sealing effect.

[0030] Further, the lifting system includes a lifting platform and a stepping motor.

[0031] The lower sealing cavity is fixed on the upper surface of the lifting platform.

[0032] By the rotation of the stepping motor to push the lifting platform to rise, the upper sealing cavity, the movable support plate and the lower sealing cavity are completely combined to form a sealed cavity.

[0033] Further, six groups of blood suction devices and blood filtration devices are respectively arranged corresponding in the housing. One blood suction device corresponds to one blood filtration device, and the recovery pipelines of the six blood filtration devices are attributed to one outlet pipeline through a distribution valve and then enter the waste liquid bottle.

[0034] Further, the range of the caliber size D of the blood suction needle and the blood inflow needle is G15≤D≤G23.

[0035] Furthermore, a plurality of slots are provided on the filter membrane, and the slots are arranged neatly and have uniform sizes.

[0036] Furthermore, a handle is provided on the outer side surface of the movable support flat plate.

[0037] As can be seen from the above technical solutions, the cell filter of the present invention is a low-pressure (which can be -0.5 to -5.0 kPa) filtering device, generating a low-pressure capture system to capture tumor cells as much as possible on the premise of not damaging / destroying the tumor cells.

[0038] Specifically, the cell filter of the present invention has the following advantages:

[0039] 1. The present invention is a low-pressure blood filtration system. By respectively providing a blood suction needle at the input end and a blood inflow needle at the output end, and the caliber of the blood suction needle is smaller than that of the blood inflow needle, the blood can be filtered with extremely low negative pressure, and the low pressure has less destructive effect on cells.

[0040] 2. The present invention does not need to pre-treat the blood and can directly perform filtration detection, ensuring the vitality of the cells. The separated live cells can be cultured for subsequent gene detection;

[0041] 3. The present invention adopts a novel membrane pore structure with consistent pore sizes and arrangement forms, avoiding the pollution of the membrane pores by white blood cells. The membrane can be in the form of a chip to increase the density of pores per unit area of the membrane, capturing tumor cells to the maximum extent, and having high efficiency and purity in capturing tumor cells.

[0042] 4. The detection of the present invention can be automated, that is, the blood suction, transportation, filtration and pipeline cleaning are all fully automated, suitable for large-scale sample detection. Brief Description of the Drawings

[0043] Figure 1 is a schematic structural diagram of the present invention;

[0044] Figure 2 is a schematic structural diagram of the blood suction device of the present invention;

[0045] Figure 3 is a schematic structural diagram of the blood filtration device of the present invention;

[0046] Figure 4 is a schematic structural diagram of the movable support flat plate of the present invention;

[0047] Figure 5 is a schematic structural diagram of the filter membrane of the present invention;

[0048] Figure 6 is a schematic structural diagram of the lifting system of the present invention;

[0049] Figure 7 It is the output pressure chart of the vacuum pump in the embodiment of the present invention;

[0050] Figure 8 It is the curve graph of the pressure difference between the pressure at the inlet end of the blood catheter and the output pressure of the vacuum pump in the embodiment of the present invention;

[0051] Figure 9 It is the structural schematic diagram of the blood recovery device in the embodiment of the present invention;

[0052] Figure 10 It is the detection experiment graph of the pressure at the inlet end of the blood inflow pipeline in the embodiment of the present invention;

[0053] Figure 11 It is the dynamic observation curve graph of the pressure drop in the sealed cavity in the embodiment of the present invention;

[0054] Figure 12 It is the schematic diagram of the working process of the present invention;

[0055] Figure 13 It is the cable layout diagram in the embodiment of the present invention;

[0056] Figure 14 It is the layout diagram of the electrical installation board in the embodiment of the present invention;

[0057] Figure 15 It is the main power circuit diagram in the embodiment of the present invention;

[0058] Figure 16 It is the N - wire and PE - wire distribution diagram in the embodiment of the present invention;

[0059] Figure 17 It is the schematic diagram of the switching power supply circuit in the embodiment of the present invention;

[0060] Figure 18 It is the 24V power distribution diagram in the embodiment of the present invention;

[0061] Figure 19 It is the PLC module circuit diagram in the embodiment of the present invention;

[0062] Figure 20 It is the extended module circuit diagram in the embodiment of the present invention;

[0063] Figure 21 It is the intermediate relay circuit diagram in the embodiment of the present invention;

[0064] Figure 22 It is the touch - screen and proximity - switch circuit diagram in the embodiment of the present invention;

[0065] Figure 23 It is the switch - valve circuit diagram in the embodiment of the present invention;

[0066] Figure 24 This is the circuit diagram of the stepping motor drive in the embodiment of the present invention;

[0067] Figure 25 This is the circuit diagram of the stepping motor in the embodiment of the present invention;

[0068] Figure 26 This is the circuit diagram of the vacuum pump control in the embodiment of the present invention;

[0069] Figure 27 This is the experimental result of lung cancer epithelial cell A549 (local) in the embodiment of the present invention. Detailed implementation manners

[0070] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are some but not all of the embodiments of the present invention.

[0071] Currently, the detection of IEST requires preprocessing of blood, using formaldehyde to fix cells, making the cells lose their vitality and unable to be used for the next-stage genomics research. The present invention does not require prior blood treatment, protects cells as much as possible, and the captured cells are live tumor cells.

[0072] As Figure 1 shown, the cell filter described in this embodiment includes a blood suction device 1, a blood filtration device 2 and a blood recovery device 3 respectively placed in a housing. The blood suction device 1 is connected to the inlet of the blood filtration device 2 through a pipeline, and the outlet of the blood filtration device 2 is connected to the blood recovery device 3 through a pipeline; the blood recovery device 3 includes a waste liquid bottle 31 and a vacuum pump 32, and the vacuum pump 32 is connected to the waste liquid bottle 31;

[0073] Among them, as Figure 2 shown, the blood suction device is used for sucking blood, and specifically includes: a blood sample test tube body 13, the mouth of the blood sample test tube body 13 is sealed, a blood suction needle 14 is arranged in the blood sample test tube body 13, the blood suction needle 14 communicates inside and outside the blood sample test tube body 13, the blood suction needle 14 is fixed on a needle attachment device 17, the needle attachment device 17 is fixed at the mouth of the test tube body 13, and the upper half of the needle attachment device 17 is sleeved with a spring 16;

[0074] Specifically, the needle attachment device 17 is as follows. The blood suction needle 14 is fixed between the upper and lower rigid splints through a special attachment device 17. The needle attachment device 17 is a hollow plastic structure with different sizes at both ends, facilitating the insertion of the needle. Its upper half fixes the needle 14 between the upper and lower splints 11, i.e., two steel plates, through the action of a spring 16. The upper and lower splints 11 are fixed to the housing by screws 12. The lower half of the needle attachment device 17 has an opening, i.e., a connecting air outlet pipe 15, except for the passage of the needle 14 through it. Its function is to maintain the pressure consistency inside and outside the blood sample tube. Only in this way can blood be sucked by the needle and transported under negative pressure.

[0075] Under the action of negative pressure, blood flows from the outlet of the blood suction needle 14 into the blood filtration device 2 through a pipeline. The inlet of the blood filtration device 2 is provided with a blood inflow needle 21, that is, blood flows into the blood filtration device 2 through the blood inflow needle 21. The calibers of the two needles, namely the blood suction needle 14 and the blood inflow needle 21, are different. Specifically, the caliber of the blood suction needle 14 is smaller than that of the blood inflow needle 21. Such a design ensures that the blood at the outflow end flows drop by drop onto the membrane and is quickly filtered through the membrane.

[0076] Regarding the transportation of blood: Blood is sucked into the plastic tube above the inlet end needle 14 under the action of negative pressure and is transferred to the blood filtration device 2 through the plastic tube. The diameter of the pipeline affects the blood suction rate.

[0077] As Figure 3 shown, the blood filtration device 2 of the embodiment of the present invention includes an upper sealing cavity 213 and a lower sealing cavity 214 corresponding up and down. A movable support plate 25 is placed between the upper sealing cavity 213 and the lower sealing cavity 214.

[0078] A sealing ring 23 is provided at the bottom of the upper sealing cavity 213. The upper sealing ring 23 is fixed on the upper cavity 22. The top of the upper sealing cavity 213 is provided with a blood inflow needle 21.

[0079] A filter hole 27 is provided at the center of the movable support plate 25, and a filter membrane 26 is provided on the filter hole 27.

[0080] A lower sealing ring 29 is provided at the top of the lower sealing cavity 214. The lower sealing ring 29 is fixed on the attachment device 28. A blood recovery pipeline 210 is provided in the lower sealing cavity 214. Blood flows out through the filter membrane 26, flows into the inlet 211 of the blood recovery pipeline, and then flows out from the outlet 212 of the blood recovery pipeline.

[0081] A plurality of slots are provided on the filter membrane 26, and the slots are arranged neatly and have uniform sizes; the slots are rectangular slots, and the length and width of the rectangular slots are 20-100 μm and 4-10 μm respectively.

[0082] Generally speaking, the sealed cavity of the blood filtration device 2 is composed of three parts. It is respectively composed of upper and lower sealed cavities and an intermediate flat plate. When there is no flat plate, only one sealed cavity is formed; when a flat plate is inserted, upper and lower cavities will be formed. The pressure of the sealed cavity is related to the vacuum pump. The pressures of the upper and lower cavities can be measured by a pressure gauge. The lower cavity is directly connected to the vacuum pump, so the pressure of the lower cavity represents the output pressure of the vacuum pump. The upper cavity is a chamber formed by the inserted flat plate and the upper sealing ring, and there are holes in the middle of the flat plate. In the case of no membrane, the pressures of the upper and lower cavities are equal. During the blood filtration process, the pore diameter of the membrane is instantaneously blocked or some of the pore diameters are blocked for a long time, resulting in a reduction in the actual permeable area of the filter membrane 26. This will cause the pressure in the upper cavity to be lower than that in the lower cavity.

[0083] After the blood flows out of the suction device 1, it passes through a pipeline and then flows into the needle 21 which is connected to the upper sealed cavity. In this way, the pressure at the inlet end of the blood pipeline can be used to reflect the pressure in the upper cavity of the sealing ring. Due to the existence of pipeline resistance, the pressure at the inlet end of the blood pipeline is lower than the pressure in the upper cavity. This pressure difference is beneficial to blood filtration. It not only slows down the blood sample suction speed but also avoids the accumulation of blood on the membrane. Thus, during the blood filtration process, the pressures in the upper and lower cavities are dynamically changing.

[0084] Specifically, the blood filtration device 2 of the embodiment of the present invention has the following characteristics:

[0085] Low pressure: Due to the very good sealing performance, liquid can be sucked from the blood tube under very low pressure conditions, and the pressure can even be lower than -0.5 kPa, and the destructive effect on tumor cells during the filtration process is relatively small.

[0086] The upper cavity 22, the lower cavity 28 and the movable support flat plate 25 are all correspondingly provided with grooves for embedding sealing rings to maintain the sealing effect. The sealing rings are arranged in the grooves, which not only ensures sealing but also does not affect the destructive effect of the liquid on the sealant.

[0087] The sealing rings in this embodiment are made of rubber and have a good sealing effect; because both the upper and lower sealing rings need to be in close contact with the flat plate during detection, the material selection is very important.

[0088] Specifically, as Figure 4As shown in the figure, a filtration hole 27 is provided at the center of the support flat plate 25, and a handle 251 is provided on the outer side surface of the support flat plate 25 for convenient taking. Specifically, the support flat plate 25 is a circular flat structure with a handle. Its structure is that there is a circular central hole at the center of the flat plate; this hole is the passage for blood filtration products, and the main red blood cells, white blood cells and other non-tumor cells in the blood flow away through this hole. At the same time, the central hole is also the passage for the formation and maintenance of negative pressure in the blood filtration system. Function: The flat plate is the carrier of the membrane, and the membrane is placed into the blood filtration system through the flat plate for the capture of tumor cells. Feature: It is very convenient to realize the circulating detection of blood samples by inserting or taking out.

[0089] As Figure 5 shown in the figure, the filtration membrane 26 of this embodiment is a material that is both acid-resistant, alkali-resistant, corrosion-resistant and has a certain hardness. There are high-density slot holes in the middle of the membrane, which are arranged neatly and have uniform sizes. Specifically, the slot holes can be rectangular slot holes, and the length and width of the rectangular slot holes are 20 - 100 μm and 4 - 10 μm respectively. The membrane holes completely cover the upper part of the middle hole of the flat plate. Under low-pressure conditions, blood is filtered through the membrane at a certain speed, and the tumor cells in the blood stay on the surface of the membrane due to their relatively large volume. After the blood filtration is completed, take out the flat plate and remove the membrane from the flat plate for the staining and identification of tumor cells. The flat plate is then used for the detection of the next-stage sample after simple cleaning. Compared with the uneven sizes and disordered arrangements of the membrane holes of the polycarbonate etched membrane, the membrane holes on the membrane chip of the present invention are of the same size and are arranged neatly.

[0090] Figure 5 P1 / P2 above: represents the sealing performance / the filtration or permeability of the membrane; V1 / V2: represents the permeability / filtration performance of the membrane, and the permeability of the membrane is mainly determined by the size and density of the membrane holes and the thickness of the membrane / the pressure difference on both sides of the membrane. The relationship between the area of the membrane holes and the size of the liquid droplets: Try to control the size of the liquid droplet volume so that the area dropped onto the membrane is smaller than the area of the membrane holes; the control of the size of the liquid droplets is mainly determined by the size of the outlet needle and the diameter of the pipeline. The diameter range of the pipeline in this embodiment of the present invention is 1 - 10 mm, and there will be no accumulation on the membrane.

[0091] At the same time, there are grooves on the upper cavity 22, and the upper sealing ring 23 is fixed in the grooves. Through the thread on its upper part and the external nut, it is fixed on the upper aluminum plate. The upper cavity 22 is fixed during the detection, and it, together with the lifting table system 4, promotes the combination and formation of the upper and lower sealing cavities.

[0092] The lower cavity 28 also has grooves on it, and the lower sealing ring 29 is fixed in the grooves. The lower cavity 28 is fixed on the upper surface flat plate of the lifting table.

[0093] As Figure 6As shown in the figure, the composition of the lifting platform system 4: This system consists of a lifting platform and a stepping motor. A lower cavity 28 is fixed on the upper surface flat plate of the lifting platform. By the rotation of the stepping motor to push the lifting of the lifting platform, the lower cavity is completely combined with the upper cavity to form a sealed cavity. The stepping motor is stimulated to rotate by a pulsed electrical signal. The stepping motor is connected to the lifting platform to push the lifting and lowering of the lifting platform, resulting in the opening and closing of the sealing ring.

[0094] Specifically, as Figure 6 shown, the lower sealing cavity 214 is fixed on the upper surface of the lifting platform 41 of the lifting platform system 4, Figure 6 The upper mark 200 is a blood recovery pipeline. The motor 43 drives the lifting bracket 44 through the motor shaft 42, and then drives the lifting platform 41 to move up and down, thereby realizing the reciprocating up and down movement of the lower sealing cavity 214.

[0095] Evaluation of the blood filtration low-pressure system in the embodiment of the present invention:

[0096] Pressure detection at the inlet end of the blood catheter: Taking a blood pipeline with a diameter of 2.0 mm as an example, test the relationship between the pressure at the inlet end of the blood catheter and the output pressure of the vacuum pump / the pressure in the lower cavity of the sealing ring under the condition of blood outflow needles with different diameters. The pressure at the inlet end of the blood catheter determines the blood sampling rate. It reflects the pressure in the upper sealing cavity and the resistance of the blood sampling pipeline.

[0097] The results are as Figure 7 and Figure 8 shown. Taking the absolute value of the negative pressure to plot the graph is convenient for observation; under the condition of the same output pressure of the vacuum pump, the larger the diameter of the blood outflow end needle, the greater the pressure at the inlet end of the blood catheter. A larger inlet end pressure means faster blood sampling. For needles with a larger diameter, it means that a relatively lower output pressure of the vacuum pump can suck blood from the end.

[0098] The explanation of the pressure difference between the upper and lower surfaces of the membrane is as follows:

[0099] The blood in the blood vessel is sucked through the continuous negative pressure action of the vacuum pump; when the membrane pores on the upper surface of the membrane are not blocked, the pressure difference ΔP between the upper and lower surfaces of the membrane is approximately 0; the pressure of the vacuum pump acts on the needle end of the blood vessel to start sucking blood;

[0100] When blood begins to drip onto the upper surface of the membrane, the cells in the blood may block the pores of the membrane (either instantaneously or over a long period); this blockage, on the one hand, increases the negative pressure in the upper cavity (the absolute value becomes smaller), resulting in a reduced ability of the needle tip to draw blood, slowing down the blood flow rate, which helps capture tumor cells on the membrane; on the other hand, the negative pressure in the lower cavity of the membrane remains unchanged (equal to the negative pressure of the vacuum pump), and this relatively enhanced negative pressure (compared to the upper cavity of the membrane) drives the blood on the membrane through the membrane, preventing the blood on the membrane from accumulating on the membrane and causing complete blockage of the membrane pores.

[0101] Once all the membrane pores are blocked, the pressure in the upper cavity of the membrane is 0, and there is no negative pressure, so blood in the blood vessel cannot be drawn anymore.

[0102] However, the filter membrane in the embodiment of the present invention ensures that even if there are cells in the membrane pores, the membrane pores will not be completely blocked, which ensures that the pressure difference between the upper and lower parts of the membrane is within a reasonable range, enabling continuous blood extraction, filtration through the membrane, and tumor cells to be left on the membrane. This design is based on the principle of low negative pressure.

[0103] It can be seen that the pressure difference between the inlet end of the blood catheter and the output pressure of the vacuum pump increases with the increase of the output pressure of the vacuum pump. This increase reflects that the influence of the needle diameter on the pressure change is very large. Thus, it can be seen the importance of selecting a suitable needle for the blood outlet end.

[0104] In summary, the blood extraction in this embodiment is achieved through the needle at the blood inlet end. The blood extraction mainly depends on the magnitude of the negative pressure and the caliber of the needle. The negative pressure is generated and regulated by the vacuum pump at the end of the filtration unit. The greater the negative pressure, the greater the blood extraction speed. When the blood extraction speed is greater than the blood filtration speed on the membrane, it will cause blood accumulation on the membrane, affecting subsequent blood filtration and tumor cell capture.

[0105] The low-pressure negative pressure is the most prominent feature of the embodiments of the present invention. In existing products, such as those of a certain company in Wuhan, all the blood is initially placed on the membrane. Once it starts working, the cells in the blood will completely occupy the membrane pores and block them. The blood and the membrane are in a sealed tube, and the pressure inside the tube is 0, so there is no pressure difference. To overcome the blockage of the membrane pores, the only thing that can be done is to increase the value of the negative pressure, and rely on a strong negative pressure to adsorb the cells from the membrane and pass through the membrane. Their pressure is as high as 400 - 600 Kpa, which is 300 times that of this embodiment. And the high pressure will cause the deformation and damage of tumor cells during the filtration process, so they need to pre-treat the blood - use formaldehyde to fix the cells and maintain the cell morphology. The disadvantage of doing this is that the obtained tumor cells are inactive; generating and maintaining a high negative pressure requires an industrial-grade vacuum pump, which is large in volume and high in cost; while the low-pressure system of this embodiment does not require pre-treatment of the blood, the required pressure is relatively low, and it will not damage the cells. The whole set of equipment is small in volume and convenient.

[0106] Finally, as Figure 9 shown, the blood recovery system 3 is realized by the negative pressure generated by the vacuum pump. The blood recovery system 3 includes a waste liquid bottle 31. After the blood is filtered by the blood filtration device 2, it flows into the waste liquid bottle 31 through the blood recovery pipeline inlet 210. The waste liquid bottle 31 is connected to the vacuum pump 33. The filtered blood enters the waste liquid bottle 31 through the pipeline. In the embodiments of the present invention, 6 blood filtration units are provided, and the recovery pipeline of each blood filtration unit passes through a distribution valve and converges to an outlet pipeline and then enters the waste liquid bottle. Regularly check the amount of waste liquid in the waste liquid bottle to prevent the waste liquid from being sucked into the vacuum pump.

[0107] At the same time, as Figure 10 shown by the experimental results, through the detection of the pressure at the inlet end of the blood inflow pipeline, it can be seen that the pressure at the inlet end of the blood inflow pipeline determines the blood suction speed and volume. This pressure is affected by three factors: the negative pressure of the vacuum pump, the diameter of the blood inflow pipeline, and the diameter of the needle at the blood outflow end. Under the premise that the diameter of the blood inflow pipeline is 2.0 mm in this experiment, the influence of the change of the vacuum pump pressure and the diameter of the needle at the blood outflow end on the pressure at the inlet end of the blood inflow pipeline was observed.

[0108] As Figure 11 shown, in order to observe the change of the pressure in the sealed cavity during the blood filtration process, a multi-channel pressure monitor is connected to the upper and lower ends of the sealed cavity. In this embodiment, 6 ml of blood is used for detection. As the blood flows in, the membrane pores are gradually blocked, and the pressure in the sealed cavity gradually increases (the negative value approaches zero). There is a pressure drop in the cavity. When the membrane pores are completely blocked, the pressure in the cavity drops to 0, and the blood in the tube cannot be extracted. After washing with PBS, the membrane pores are gradually flushed open, and the negative pressure in the cavity reappears, and the pressure drop becomes lower.

[0109] The embodiment of the present invention has 6 blood filtration units, each unit being independent; more filtration units can also be connected in parallel, and more samples will be detected in one test. The whole process is divided into three parts: blood aspiration, blood filtration, and blood recovery, as Figure 12 shown.

[0110] The device corresponding to the embodiment of the present invention specifically adopts automatic control, and the relevant control circuit is as Figures 13 - 26 shown. When in use, the present invention starts the machine according to the instructions in the manual. Select the "detection" program on the display screen and input the number of blood samples to be tested. Place the membrane on the flat plate and insert it into the inverted "T"-shaped hole, and insert the tube containing the blood sample into the needle device. Press the start detection key, and the instrument starts to work. Blood is aspirated from the end of the needle and transported to the blood filtration unit through the blood catheter under the action of negative pressure. When the blood passes through the filtration membrane on the flat plate, abnormal cells are left on the membrane. After the blood aspiration is completed, the detection program enters the cleaning process, and the needle, blood transfer pipeline, blood filtration unit, and blood recovery pipeline are automatically cleaned. The purpose of cleaning is to detect the next batch of blood samples, and this process is repeated until all blood samples are detected.

[0111] The following are the specific experiments applying this embodiment:

[0112] Experiment 1: Detection of the retention rate (spiked-in) of tumor cells

[0113] 1. Take 200 μl of lung cancer epithelial cells A549 at 2.4x106 / ml, add 100 μl of 2 mM Calcein-AM, and stain at room temperature for 15 minutes. Centrifuge at 200 g for 5 minutes, pour off the supernatant, and wash the cells twice with PBS to remove excess dye. Then, resuspend the cells in PBS containing 2 mM EDTA and 0.5% BSA.

[0114] 2. Through statistical sampling by serial dilution, dilute the cells to an ideal concentration of 10 - 100 cells / 50 μL. Subsequently, use a micropipette to transfer 50 μL of the diluted cell suspension to a 96-well microtiter plate, and count the number of cells in the transferred suspension under a microscope. Repeat the counting three times to determine the actual number of cells in the suspension. After repeating these operations, adjust the volume of the cell suspension added to 1 ml of whole blood to prepare a tumor cell spiked sample.

[0115] 3. Add a certain amount of tumor cells to 1 ml of healthy human whole blood and mix well. The blood is tested on the machine; the size of the membrane pores in this experiment is 6x40 μM, the total area of the membrane pores is 6x6 mm, the pressure of the vacuum pump is 2.0 ± 0.1 Kpa, and the flow rate is 500 μl / min.

[0116] 4. Counting of viable tumor cells on the membrane. Since Calcein-AM only binds to the cell nuclei of viable cells and emits green light under the excitation of blue light in a fluorescence microscope, it can be observed and counted.

[0117] Experimental results:

[0118] As Figure 27 shown, under a 10x objective lens, the number of captured tumor cells can be obtained by observing and counting the number of green fluorescent cells in the microscope field of view. The capture rate of tumor cell A549 is 92 ± 3%.

[0119] Experiment 2: Capture of CTCs in the peripheral blood of tumor patients

[0120] 1. For 11 patients with various malignant tumors, 3 ml of peripheral blood of the patients was drawn with an EDTA anticoagulant tube and diluted to 6 ml with PBS at a ratio of 1:1.

[0121] 2. The diluted blood was loaded onto the machine for filtration. The pore size of the membrane was 6x40 uM, the pressure of the vacuum pump was 2.0 ± 0.1 Kpa, and the flow rate was 500 uL / min.

[0122] 3. After the blood filtration was completed, the membrane was taken out, stained with Wright's staining method, and circulating tumor cells (CTCs) were identified and counted.

[0123] 4. Criteria for CTC determination: a. Abnormal cell shape and uneven size; b. Nucleus diameter greater than 24 um; c. Nucleus atypia; d. High nuclear-cytoplasmic ratio (nucleus / cytoplasm); e. Thickened and irregular nuclear membrane with a three-dimensional sense. (Those meeting the above 4 criteria are determined to be malignant tumor cells);

[0124] The experimental results are as follows:

[0125]

[0126] To sum up, the present invention is based on the principle that the volume of tumor cells is larger than that of normal blood cells. During blood filtration, they are retained on the surface of the membrane because they cannot pass through the pore size of the membrane chip on the membrane, and thus are captured. This capture is ① under low-pressure conditions, with very little damage to tumor cells, and very little damage to cells by shear force during blood flow; it protects tumor cells as much as possible; ② no pre-treatment of blood is required: currently, the ISET method used in China requires pre-treatment of blood, mainly by treating blood cells with about 1% formaldehyde solution to fix cell membranes and cell structures. The captured cells are dead cells and cannot be used for subsequent tests such as cell culture. ③ Viable tumor cells are provided. ④ Fully automated and highly efficient in capturing tumor cells, suitable for large-scale sample detection.

[0127] Relatively speaking, the embodiments of the present invention have the following advantages:

[0128] 1. Detection under low pressure:

[0129] Currently, the main problem faced by ISET is how to capture live tumor cells in blood under low pressure conditions as much as possible. The current domestic technology captures cells from the membrane by applying increased negative pressure. High negative pressure requires an industrial-grade vacuum pump. The industrial-grade vacuum pump increases capital investment on the one hand, and at the same time generates high noise and large volume, making the entire set of equipment very large. The change in blood shear force generated under high negative pressure will also damage blood cells and tumor cells. How to capture tumor cells under very low pressure conditions is a key issue. Low pressure (-1.0 to -5.0 kPa) protects cells on the one hand and miniaturizes the entire device on the other hand.

[0130] 2. Blood does not require pretreatment; fewer steps and less damage to tumor cells.

[0131] 3. Practical and convenient: The low pressure is achieved through the unique filtering unit of this embodiment, which is both convenient and practical. The entire system is quickly realized through steps such as the quick opening and closing of the upper and lower sealing cavities pushed by the lifting platform and the insertion and removal of the flat plate / membrane. It is very suitable for large-scale sample detection.

[0132] 4. Low cost: Only the membrane is a consumable;

[0133] 5. The detection process, including filtration and cleaning, is fully automated and precisely controlled.

[0134] All in all, the advantages of the embodiments of the present invention in terms of filtration results are as follows:

[0135] a). High capture efficiency;

[0136] b). Live tumor cells;

[0137] c). The cells can be used for subsequent genetic testing;

[0138] d). Large-scale sample detection: Extremely short sample processing process and fast detection process.

[0139] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A cell filter, comprising a housing, characterized in that: The device also includes a blood suction device (1), a blood filtering device (2), and a blood recovery device (3) respectively disposed in the housing. The blood recovery device (3) includes a waste liquid bottle (31) and a vacuum pump (32), and the vacuum pump (32) is connected to the waste liquid bottle (31). The blood suction device (1) is in sealed communication with the inlet of the blood filtering device (2) through a pipeline, and the outlet of the blood filtering device (2) is in sealed communication with the waste liquid bottle (31) of the blood recovery device (3) through a pipeline; Wherein, a blood suction needle (14) is provided in the blood suction device (1); The blood filtering device (2) is provided with an upper sealed chamber (213), a movable supporting plate (25) and a lower sealed chamber (214) in sequence from bottom to bottom, and the lower sealed chamber (214) is fixed on the lifting system; The upper sealing cavity (213) comprises an upper cavity (22) and an upper sealing ring (23) fixed to the bottom of the upper cavity (22), and the top of the upper sealing cavity (213) is sealed to connect blood to the needle (21); The movable support plate (25) is placed between the upper sealed chamber (213) and the lower sealed chamber (214). By starting the lifting system to move up and down, the lower sealed chamber (214) and the movable support plate (25) can be separated from and closed to the upper sealed chamber (213) in sequence. A filter hole (27) is provided at the center of the movable supporting plate (25), and a filter membrane (26) is provided on the filter hole (27); a plurality of slots are provided on the filter membrane (26), and the slots are arranged neatly and have uniform sizes; The lower sealed cavity (214) includes a lower cavity (28) and a lower sealing ring (29) fixed on the top of the lower cavity (28), and the lower sealed cavity (214) is sealed and connected to the blood recovery pipeline inlet (210); The blood passes through the blood suction needle (14) of the blood suction device (1) to the blood flow needle (21) of the blood filtering device (2), then drips onto the filter membrane (26) and flows out through the blood recovery pipeline outlet (212) into the waste liquid bottle (31); The caliber of the blood suction needle (14) is smaller than the caliber of the blood inflow needle (21); The blood suction device (1) comprises a blood sample test tube body (13), the tube mouth of the blood sample test tube body (13) is sealed, a blood suction needle (14) is arranged in the blood sample test tube body (13), and the blood suction needle (14) is connected to the inside and outside of the blood sample test tube body (13); The blood aspirating needle (14) is fixed on a needle attachment device (17), and the needle attachment device (17) is fixed at the tube mouth of the blood sample test tube body (13); the blood aspirating needle (14) is fixed between the upper and lower rigid splints through the needle attachment device (17), and the upper and lower rigid splints are fixed on the housing; The needle attachment device (17) is a hollow plastic structure with two ends of different sizes; the blood suction needle (14) passes through the needle attachment device (17); a spring is set on the upper half of the needle attachment device (17), and the needle is fixed between the two steel plates through the spring.

2. The cell filter according to claim 1, wherein: An air outlet pipe (15) is also provided on the lower half of the needle attachment device (17).

3. The cell filter according to claim 1, wherein: The upper cavity (22), the lower cavity (28) and the movable support plate (25) are respectively provided with grooves for embedding sealing rings to maintain the sealing effect.

4. The cell filter according to claim 1, wherein: The lifting system includes a lifting platform and a stepping motor; A lower sealing cavity (214) is fixed on the upper surface of the lifting platform; By rotating the stepping motor to push the lifting platform to rise, the upper sealing cavity (213), the movable support plate (25) and the lower sealing cavity (214) are completely combined to form a sealed cavity.

5. The cell filter according to claim 1, characterized in that: Six groups of blood suction devices (1) and blood filtration devices (2) are respectively arranged in the shell. One blood suction device (1) corresponds to one blood filtration device (2). The recovery pipelines of the six blood filtration devices (2) pass through a distribution valve and are collected into an outlet pipeline and then enter the waste liquid bottle (31).

6. The cell filter according to claim 1, wherein: The caliber D of the blood suction needle (14) and the blood inflow needle (21) ranges from G15≤D≤G23.

7. The cell filter according to claim 1, characterized in that: The slot hole is a rectangular slot hole, and the length and width of the rectangular slot hole are 20-100 μm and 4-10 μm respectively.

8. The cell filter according to claim 1, wherein: A handle (251) is arranged on the outer side surface of the movable support plate (25).

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