WO2014086302A1 - 一种用于生物样本体外纯化处理的套管 - Google Patents

一种用于生物样本体外纯化处理的套管 Download PDF

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Publication number
WO2014086302A1
WO2014086302A1 PCT/CN2013/088668 CN2013088668W WO2014086302A1 WO 2014086302 A1 WO2014086302 A1 WO 2014086302A1 CN 2013088668 W CN2013088668 W CN 2013088668W WO 2014086302 A1 WO2014086302 A1 WO 2014086302A1
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WO
WIPO (PCT)
Prior art keywords
movable baffle
movable
casing
sealing ring
polygonal
Prior art date
Application number
PCT/CN2013/088668
Other languages
English (en)
French (fr)
Inventor
赵侃
王春有
王冬明
刘湘连
甘朝
Original Assignee
北京东方华辉生物医药科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from CN201210518450.0A external-priority patent/CN103849562B/zh
Priority claimed from CN 201220708928 external-priority patent/CN202968559U/zh
Priority claimed from CN 201220704864 external-priority patent/CN202968558U/zh
Application filed by 北京东方华辉生物医药科技有限公司 filed Critical 北京东方华辉生物医药科技有限公司
Publication of WO2014086302A1 publication Critical patent/WO2014086302A1/zh

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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5021—Test tubes specially adapted for centrifugation purposes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0609—Holders integrated in container to position an object
    • B01L2300/0618—Holders integrated in container to position an object for removable separation walls
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0681—Filter

Definitions

  • the invention relates to a sleeve for in vitro purification treatment of a biological sample, and belongs to the technical field of biomedicine. Background technique
  • Enrichment of biological particles in biological samples by centrifugation is one of the most widely used techniques in biological research.
  • Many biological particles can be enriched by the technique of centrifugation, for example, animal or plant cells, subcellular structures, bacteria, viruses or biological macromolecules.
  • the performance of these biological particles in the suspension under centrifugation depends mainly on the size and density of the biological particles, which can be explained by the Stokes equation.
  • the Stokes equation (see the following equation) describes the variation of the rate of motion of spherical particles when subjected to centrifugation in a liquid medium:
  • d represents the diameter of the spherical particles
  • p 2 represents the density of the spherical particles
  • n represents the viscosity of the liquid medium
  • g represents the centrifugal force.
  • the lymphocytes in a human blood sample are enriched in vitro by centrifugation, since the density of human lymphocytes is about 1.077 g/cm 3 , it can be realized from a human blood sample by using a density medium having a density of 1.077 g/cm 3 .
  • a density medium having a density of 1.077 g/cm 3 .
  • a material such as barium chloride or barium sulfate which can be formulated into a solution of the density range should be selected as the density medium formulation.
  • the in vitro purification treatment of the biological sample by the principle of centrifugation that is, the step of enriching the biological particles from the biological sample is substantially similar.
  • the general operation steps include: (1) pre-dilution of blood samples with a certain proportion of balanced salt solution or physiological saline; (2) dilution of the blood samples; The blood sample is carefully placed in the centrifuge vessel pre-filled with the density medium along the wall of the centrifuge vessel to keep the interface between the sample and the medium clear and no mixing occurs.
  • This process is a key step to ensure the enrichment effect, often referred to as "blood deposition.”
  • step (2) the operator must carefully deposit the diluted blood sample on the density medium to keep the interface between the sample and the medium clear and no mixing occurs. This is because, if the blood sample is mixed with the density medium, the "white film layer" that is finally enriched between the sample and the dielectric layer will become very loose and contain a lot of red blood cell contamination, which will not only be given in step (4).
  • the collection operation brings obstacles in visual judgment and also has a serious adverse effect on the yield and purity of the target cells.
  • the operation of "blooming" is characterized by time-consuming and laborious measures, and the technical requirements of the operator are high, which is not conducive to achieving large-scale treatment, especially when used for clinical therapeutic purposes, the large-scale treatment of a large number of blood samples is A major technical problem.
  • the operator first needs to accurately judge the position of the "white film layer", and then use a Pasteur pipette or a pipette to extend into the liquid surface to absorb the enrichment.
  • centrifuge tubes specifically designed to purify lymphocytes from blood samples, including Leucosep® centrifuge tubes developed by Greiner Bio-one, Germany, which are characterized by a lower portion of the tube.
  • a porous filter barrier is installed and the centrifuge tube is divided into upper and lower compartments. Since the pore size of the barrier is relatively small, the blood sample cannot pass through the barrier immediately, so that the blood sample can be directly poured into the centrifuge tube in which the lower compartment is pre-filled with the density medium without worrying about the mixing of the blood sample with the density medium.
  • the defects of such a centrifuge tube having a porous filter barrier structure are also obvious, and specifically include the following points: (a) Since the pore size of the barrier is relatively small, not only the blood sample cannot pass through the barrier immediately, but also the density medium.
  • the barrier can not immediately pass through the barrier, such a device is therefore suitable for standard operation they are first upper compartment was added an appropriate amount of medium density, such as Ficoll-Paque TM Plus, by centrifugation minutes, the density of the medium through all of the barrier into the lower In the compartment, a subsequent "blooding" operation can be performed, which again introduces additional trouble to the separation of the blood sample.
  • the amount of density medium added needs to be very accurately controlled, which should be slightly lower than the height of the barrier. If the amount added is too small, the purification effect will be adversely affected; if the amount added is too large, it needs to be recycled more than The density medium of the barrier height, which also brings inconvenience to the separation operation.
  • each centrifuge tube having a porous filter barrier structure is about several tens to one hundred times the price of the same capacity centrifuge tube, which greatly limits the products of this type. widely used.
  • Chinese Patent 200610063125.4 provides a lymphocyte separation tube provided with a porous porous material at the bottom of the tube, which can adsorb the density medium into a semi-solid state, and can directly pour blood samples into the blood sample during purification.
  • the separation tube does not have to worry about mixing the interface between the blood and the separation liquid, which solves the problem that the "blooding" operation is time consuming and laborious.
  • the loose porous material and the tube body can be fixed by friction, the technique does not require the use of an adhesive, and the process difficulty is greatly reduced, thereby overcoming the above defects (b) and (c). The problem described in ).
  • lymphocyte separation tube provided with a porous porous material at the bottom of the tube still faces the time-consuming problem of the density medium addition process. Since the porous material can prevent the blood from passing through immediately, the adsorption and penetration of the density medium also takes a certain time. It needs to be realized by "dropping"; and, since the porous material itself is not transparent, the amount of density medium added is also difficult to achieve accurate control, which in turn leads to control of the separation effect of the blood sample and reproducibility of the result. It is very difficult.
  • the above patent also provides another product form in which a lymphocyte separation liquid (density medium) is previously added to the porous porous material of the tube body, which can achieve accurate control of "out of the box” and the amount of density medium added.
  • an object of the present invention is to provide a cannula for in vitro purification treatment of a biological sample, which can be placed in a centrifuge container for in vitro purification treatment of a biological sample, the cannula having It is convenient and practical, simple in production process, low in cost, safe and non-toxic, and is especially suitable for large-scale processing of biological samples, which greatly simplifies the operation process.
  • the present invention first provides a cannula for in vitro purification treatment of a biological sample, which comprises a tube body, a plurality of movable baffles, a connecting shaft, a sealing ring, and a coaxial locking switch, wherein:
  • the tube body has a cylindrical shape (preferably a cylindrical shape), and has an outwardly facing flange at an upper end thereof, and a plurality of first openings are formed on a bottom end surface thereof, and a plurality of second openings and a plurality of bottom ends of the side walls are provided An outwardly protruding bump, wherein the bump is located above a connecting portion of two adjacent second openings;
  • the sealing ring is sleeved on an outer side of the second cornice
  • the movable baffle is disposed at a bottom of the pipe body, and the movable baffle is provided with a plurality of third openings, and an outer edge of the movable baffle is in one-to-one correspondence with the second opening and is sealed with the Circle contact
  • the connecting shaft is located at a central position of the pipe body, the top end thereof is connected to the coaxial locking switch, and the bottom end is provided with a polygonal member, and each side of the polygonal member is one by one with the inner edge of one of the movable baffles Corresponding and contacting;
  • the coaxial locking switch is located on an upper surface of the flange of the tubular body and is rotatable along the upper surface; when the movable shutter is in an open state, the first opening and the third opening At least partially overlapping; when the movable baffle is in a closed state, there is no overlapping portion between the first opening and the third opening, and the shape and size of the second opening is opposite to the movable baffle The outer edges match.
  • the above-described cannula provided by the present invention is for use in a centrifuge container (e.g., a conventional centrifuge tube, a centrifuge bottle) to achieve enrichment of biological particles in a biological sample.
  • a centrifuge container e.g., a conventional centrifuge tube, a centrifuge bottle
  • the outer diameter of the sleeve body is slightly smaller than the inner diameter of the centrifuge container, and the two are actively fitted, and the length (or height) of the sleeve can be as needed. Determined by the size of the centrifuge vessel.
  • the flange of the top of the sleeve is sized to enable the sleeve to be hung on the centrifuge container for fixation, and does not affect the use of the centrifuge container lid, preferably equal to or slightly smaller than the centrifuge volume
  • the outer diameter of the thread The transverse dimension (outer diameter or height relative to the outer wall of the tube) of the projection located in the lower part of the casing tube should be such that the projection can be in close contact with the inner wall of the centrifuge vessel and the casing can be placed smoothly into the centrifuge vessel. Neutralization is taken out.
  • the diameter of the circle formed by the outer edge of the bump is the same as or slightly smaller than the inner diameter of the centrifuge container.
  • the bottom surface of the bump should be in contact with the sealing ring so as to be able to press the sealing ring to avoid the upward tilting thereof, and at the same time, when the movable baffle is in the open state, the convex block can also press the outer edge of the movable baffle to avoid It is also upturned, and it can also seal a part of the gap generated after the movable baffle is opened together with the rubber ring.
  • the movable baffle is mounted at the bottom of the pipe body and is free to move in a certain range in the radial direction.
  • the outer edges of the movable baffles are curved and connected together to form a circle.
  • Each of the movable baffles is provided with a number of third openings for the passage of biological particles in the biological sample.
  • the movable baffle has two states of opening and closing.
  • the sleeve provided by the invention comprises 3-12 (preferably 3-6) flaps, the number of second openings, the number of bumps, the number of sides of the polygonal part and The number of movable baffles is the same.
  • the polygonal part at the bottom of the connecting shaft can be an equilateral triangle-positive dodecagonal, corresponding to 3-6 movable baffles, and the polygonal part at the bottom of the connecting shaft can be an equilateral triangle - positive hexagonal shape.
  • the movable baffle is a ring-shaped fan, and the thickness of the movable baffle is the same as the thickness of the polygonal member, and all the movable baffles and the polygonal member form a complete disc shape; or the movable block
  • the plate is fan-shaped, the thickness of the central portion of the baffle is lower than the thickness of the outer portion, and the sum of the thickness of the central portion of the movable baffle and the thickness of the polygonal member is equal to or greater than the thickness of the outer portion of the movable baffle, all the movable baffles Forms a complete disc shape with polygonal parts.
  • the movable baffle When the movable baffle is ring-shaped, its inner edge can be straight, and all the movable baffles form a disc with a polygonal hole in the middle, and the polygonal hole has the same shape as the polygonal part at the bottom end of the connecting shaft;
  • the central portion In the fan shape, the central portion is thinner than the outer half, and the central portions of all the movable baffles are combined to form a disc having a groove in the middle, and the side of the groove is preferably linear, and the shape and the polygonal member can Cooperating, the polygonal part is also slightly thinner to match the thickness of the movable baffle.
  • the outer rim side of the movable baffle has a recess that cooperates with the sealing ring.
  • the sealing ring can be placed in the groove to maintain firmness to prevent slippage.
  • the bottom end surface of the pipe body is provided with a first through hole
  • the movable baffle has a fan shape with an arcuate notch at the center of the baffle, the central portion of the baffle has a thickness lower than the thickness of the outer portion, and the sum of the thickness of the central portion of the movable baffle and the thickness of the polygonal member Equal to or greater than the thickness of the outer portion of the movable baffle, all the movable baffles and the polygonal member form a complete disc shape, and the arcuate notches of all the movable baffles are combined to form a first and the first a second through hole corresponding to the through hole; a bottom portion of the polygonal member has a columnar member, the column member passes through the second through hole and the first through hole, and the end of the column member is a latch The latching end longitudinally fixes the connecting shaft and the polygonal member.
  • the first through hole is used to pass the columnar member at the bottom of the polygonal member, and the locking end of the end of the column member is located below the bottom end surface, which can longitudinally fix the polygonal member and the connecting shaft to prevent the occurrence thereof.
  • Undesirable longitudinal displacement which prevents the flapper from being tilted up during rotation and longitudinal displacement of the connecting shaft as it rotates, resulting in unintended displacement of the flapper.
  • the columnar member provided at the bottom of the polygonal member is preferably disposed at a center position of the bottom portion of the polygonal member, along an extension line direction of the center line of the connecting shaft.
  • the middle portion of the columnar member is thinner, and the locking end of the end portion may be any three-dimensional shape for realizing the locking function, preferably a spherical shape, a rectangular parallelepiped shape, a polyhedral shape, or the like, as long as the size thereof can be larger than the first through hole and thus stuck in the first
  • the polygonal member and the connecting shaft are longitudinally fixed at the through hole.
  • the horizontal size of the locking end is larger than the diameter of the first through hole.
  • a first through hole is formed in the bottom end surface of the tubular body of the sleeve for passing the columnar member at the bottom of the polygonal member, and the locking end of the end of the column member is located below the bottom end surface, which can longitudinally connect the polygonal member and the connecting shaft It is fixed on the casing tube to prevent unwanted longitudinal displacement, which can prevent the movable baffle from being upturned during rotation and avoid longitudinal displacement of the connecting shaft during rotation, avoiding the unexpected movement of the movable baffle Displacement.
  • the inner edge of the flap is provided with a notch that matches the angle of the polygonal member.
  • the notch on the movable baffle is provided on the inner edge which is in contact with the side of the polygonal member.
  • the above-mentioned groove is oriented toward the center, and its shape may be any shape capable of embedding a corner of the polygonal member therein to perform a rotational locking effect, such as a semicircle, a triangle, preferably a triangle.
  • the first opening is provided on the bottom end face of the tubular body of the cannula for allowing biological particles (e.g., blood cells) in the biological sample to pass freely in the centrifugal field and in contact with the density medium contained in the centrifuge container.
  • biological particles e.g., blood cells
  • the shape and size of the first opening can be selected as needed, but adapted to the third opening on the movable baffle so as to be hit.
  • the open state enables the biological sample to contact the density medium through the coincident portion of the first opening and the third opening.
  • the first opening and the third opening at least partially overlap (preferably completely coincident, in this case, the first opening of the corresponding position and The third opening may have the same shape); when the movable shutter is in the closed state, there is no overlapping portion between the first opening and the third opening, that is, closed to each other.
  • the second opening provided on the side wall of the sleeve may be arranged as needed, the number of which shall be the same as the number of the movable baffle, and the second opening shall allow the movable baffle to freely enter and satisfy when in the open state
  • the closing requirement that is, the size and shape of the second opening should match the size and shape of the outer edge of the movable baffle.
  • the number of bumps provided on the side wall of the pipe body is also the same as the number of movable baffles.
  • the second opening is disposed along the outer wall of the sleeve, and may be a rectangular shape (or a rectangular shape of the curved surface), that is, the upper and lower sides are respectively parallel to the bottom end surface of the sleeve, and the left and right sides are respectively perpendicular to the bottom end surface of the sleeve.
  • the sealing ring is sleeved outside the second opening to seal the gap between the sleeve and the centrifuge container during use.
  • the thickness of the sealing ring is the same as the height of the second opening.
  • the sealing ring can be any ring that enables sealing, preferably a resilient sealing ring, such as a rubber ring.
  • the inner diameter of the sealing ring can be the same as the outer diameter of the tubular body to facilitate the sleeve.
  • the inner edge of the sealing ring is in contact with the outer edge of the movable baffle, and the diameter of the circle formed by the outer edges of the respective movable baffles is the same as the inner diameter of the sealing ring.
  • the movable baffle when the movable baffle is in the closed state, it is in the "contracted” state, and the sealing ring is also in the “contracted” state (the state without elastic deformation), and the outer diameter is slightly smaller than the inner diameter of the centrifugal container.
  • the gap between the casing tube and the centrifugal container is not sealed, and the casing tube can be freely taken out or placed from the centrifugal container.
  • the third opening on the movable baffle and the first opening at the bottom of the tube body are mutually blocked by the phase difference, the tube body is in a relatively closed state, and the biological sample cannot pass; and when the movable shutter is in the open state, it is in the "expanded” state, the third opening U on the movable baffle and the bottom of the tube body
  • the first open Us at least partially coincide with each other, and the biological sample can pass freely.
  • the sealing ring is pushed outward by the edge of the movable baffle along the expansion of the movable baffle to become an "expanded" state.
  • the polygonal member connecting the bottom end of the shaft is in close contact with the center side of the movable shutter, and the state switching (opening and closing) of the movable shutter is realized by the rotation of the polygon member, the connecting shaft and the coaxial locking switch.
  • the polygonal part can also have two states of use, open and closed (open and closed here) The description is defined to be consistent with the state of the movable baffle, and does not mean that the shape of the polygonal member itself is to be opened or closed.
  • the edge of the polygonal member and the center side of the movable baffle The edges are in parallel contact, the movable baffle is in a closed state, the sealing ring is contracted, and the tubular body is in a relatively closed state; when the polygonal member is in an open state, the apex of the polygonal member is in contact with the central side edge of the movable baffle, and the movable baffle is in a radial direction A displacement is generated outwardly, and the third opening on the movable baffle and the first opening at the bottom of the tubular body at least partially coincide with each other, from the closed state to the open state.
  • a sleeve having a notch matching the angle of the polygonal member is provided for the inner edge of the movable baffle, and the edge of the polygonal member is opposite to the movable baffle when the polygonal member is in the closed state
  • the central side edges are in parallel contact, the movable baffle is in a closed state, the sealing ring is contracted, and the tubular body is in a relatively closed state; when the coaxial locking switch is rotated, the connecting shaft and the polygonal member are also rotated, in the process, the top of the polygonal member
  • the corner pushes the movable baffle outwardly until the coaxial locking switch is turned to the predetermined position, and the polygonal member reaches the open state; when the polygonal member is in the open state, the top corner of the polygonal member is embedded on the center side edge of the movable baffle In the notch, the movable baffle is displaced outward in the radial direction, and the third opening
  • each of the apex angles of the polygonal member is fitted into the corresponding notch, and the polygonal member can be locked in the rotational direction in the case of handling or centrifugation without applying an external force in the rotational direction.
  • This notch (along with the coaxial locking switch) ensures that the movable baffle does not move unintentionally during use, thus ensuring good stability of the bushing.
  • the connecting shaft is located at the center of the pipe body, coincides with the central axis of the pipe body, and its top end is connected to the coaxial lock switch.
  • the coaxial locking switch can rotate the connecting shaft to rotate, thereby changing the position state of the polygonal member, thereby switching the movable shutter between the closed state and the open state.
  • the coaxial locking switch can realize the positional locking of the annular flange at the upper end of the pipe body through a certain locking structure, and avoid the movable baffle in the centrifugal field or the carrying state. Unexpected displacement occurs underneath.
  • the locking structure may be any device capable of achieving the above locking.
  • the flange of the sleeve is provided with at least two pairs (four) of locking grooves, the locking grooves are opposite to each other, and the coaxial locking switch comprises a cross bar.
  • the middle of the cross bar is connected with the top end of the connecting shaft and can drive the connecting shaft to rotate.
  • the two ends of the cross bar are respectively located in a pair of locking grooves and can be switched between different locking grooves.
  • four locking grooves are arranged on the flange of the sleeve so as to oppose each other.
  • the middle of the crossbar of the coaxial locking structure is connected with the top end of the connecting shaft and can drive the connecting shaft to rotate.
  • the cross bar can be rotated into the next pair of locking grooves by an external force, and the rotation of the movable baffle between the open and closed states can be realized by the rotation.
  • the shape of the crossbar is only suitable for the shape of the locking groove.
  • the size of the crossbar is equivalent to the size of the locking groove, so that the crossbar has a certain resistance when it falls into the locking groove, and the crossbar is secured by the resistance in the longitudinal direction.
  • the two ends of the cross bar may include two upward heads, so as to apply an external force to rotate the cross bar and apply two forces directed to the center of the cross bar to lift the two ends of the cross bar away from the pair of locking slots.
  • the baffle is tilted up when it is rotated.
  • a force directed to the center acts on both ends of the crossbar and reaches a certain size, the crossbar can be temporarily disengaged from the locking groove and transferred to the other pair of locking slots to be locked.
  • the crossbar preferably has a certain Elastic, so as to be turned in and out; the distance between the two pairs of locking grooves can be determined according to the number of movable baffles, as long as the transition between the open and closed states of the movable baffle and the locking can be achieved.
  • the material used for the sleeve provided by the present invention may be any medical material that meets relevant sanitary standards.
  • the material of the sleeve may be a plastic material for preparing a syringe for clinical injection, and the rubber of the rubber ring used for sealing.
  • the material may be a medical rubber that meets hygienic standards, such as a rubber material for making a rubber part of a front portion of a syringe push rod for clinical injection, such as medical silicone rubber or the like.
  • the present invention also provides a centrifuge container for in vitro purification treatment of a biological sample, comprising the above-mentioned cannula and centrifuge container body for in vitro purification treatment of a biological sample, wherein the cannula is located in the centrifuge container body and both are active Matching.
  • the above-mentioned centrifugal container may be a centrifuge tube or a centrifuge bottle or the like.
  • the present invention also provides a method for in vitro purification treatment of a biological sample, which comprises enriching biological particles in a biological sample using the above-described centrifugal container, comprising the steps of:
  • the movable baffle of the casing is closed, the casing is taken out from the centrifuge vessel body, and the bioparticle enrichment layer is transferred to complete the enrichment of the biological particles in the biological sample.
  • the bioparticle-rich layer separated by the above method can be used for different purposes by appropriate treatment, and the specific treatment can be carried out according to a conventional manner.
  • the amount of the density medium to be charged may be determined as needed.
  • the upper surface of the density medium loaded into the body of the centrifuge container is in contact with or slightly higher than the bottom end surface of the sleeve.
  • the biological sample is a blood or a tissue sample
  • the biological particles in the biological sample are lymphocytes, monocytes, hematopoietic stem cells, and interstitial cells. Stem cells, etc.
  • the cannula provided by the present invention is proposed for the difficulty and problem of enriching biological particles from biological samples such as blood or tissue samples by density gradient centrifugation, and can solve the "blooding" operation and "absorbing the white film layer". "Operation is time consuming and unfavorable for the problem of large-scale processing of biological samples; it is also possible to solve the defects described in (a) - (e) existing in the prior art products or technologies provided in the background art.
  • the sleeve provided by the invention is a device which is convenient and practical, has low production cost, simple production process and is safe and non-toxic for in vitro purification treatment of biological samples.
  • FIG. 1 is a schematic view showing the overall structure of a sleeve provided in Embodiment 1;
  • FIG. 2 is a schematic exploded view of the movable baffle and the connecting shaft of the sleeve provided in Embodiment 1;
  • FIG. 3 is a schematic view showing a closed state of a movable baffle of the sleeve provided in Embodiment 1;
  • FIG. 4 is a schematic view showing the open state of the movable baffle of the sleeve provided in Embodiment 1;
  • Figure 5 is a schematic view showing the first opening of the bottom end surface of the sleeve provided in Embodiment 1;
  • FIG. 6 is a schematic view showing a second jaw and a bump on the side of the sleeve provided in Embodiment 1;
  • FIG. 7 is a schematic structural view of a movable shutter and a polygonal member in Embodiment 2;
  • FIG. 8 is a schematic view showing a setting manner of a coaxial locking switch in Embodiment 1;
  • Figure 9 is a cross-sectional view taken along line A-A of Figure 8.
  • Figure 10 is a schematic exploded view of the movable baffle and the connecting shaft of the bushing provided in Embodiment 3;
  • Figure 11 is a schematic view showing the first opening and the first through hole of the bottom end surface of the sleeve provided in Embodiment 3;
  • Figure 12 is a schematic view of a single movable baffle provided in Embodiment 3.
  • FIG. 13 is a schematic view showing a second through hole composed of a curved notch of the movable baffle provided in Embodiment 3;
  • FIG. 14 is a schematic structural view of the movable baffle and the polygonal member in Embodiment 3;
  • Figure 15 is a schematic view showing the closed state of the movable shutter of the sleeve provided in Embodiment 4.
  • Figure 16 is a schematic view of the movable baffle with a notch provided in Embodiment 4.
  • Figure 17 is a schematic view of the movable baffle with a notch provided in Embodiment 5;
  • Figure 18 is a schematic view showing the open state of the movable shutter of the sleeve provided in Embodiment 4.
  • FIG. 19 is a schematic flow chart of a method for in vitro purification treatment of a biological sample provided in Example 7.
  • the embodiment provides a sleeve for in vitro purification treatment of a biological sample, which comprises a tube body 1, six movable baffles 2, a connecting shaft 3, a sealing ring 4, and a coaxial locking switch 5, as shown in FIG. , among them:
  • the upper end of the tubular body 1 has an outwardly facing flange 6, and the bottom end surface of the tubular body 1 is provided with a plurality of first openings 7 (as shown in FIG. 5), and the bottom end of the side wall is provided with a plurality of second openings 9 and a plurality of outwardly protruding bumps 8 , wherein the bumps 8 are located above the connecting portion 12 of the adjacent two second openings 9 (as shown in FIG. 6 );
  • the sealing ring 4 is a rubber ring which is sleeved on the outer side of the second opening 9;
  • the movable baffle 2 is disposed at the bottom of the pipe body 1, which is a ring-shaped fan, and is provided with a plurality of third openings 10, and the outer edge of the movable baffle 2 is in one-to-one correspondence with the second port 9 and the sealing ring 4 Contact (as shown in Figure 1, Figure 2, Figure 3, Figure 4);
  • the connecting shaft 3 is located at the center of the pipe body 1, the top end of which is connected to the coaxial locking switch 5, and the bottom end is provided with a polygonal member 11 (in this embodiment) In the example, a regular hexagon), each side of the polygonal member 11 is in one-to-one correspondence with the inner edge of a movable shutter 2 (as shown in FIGS. 2 and 3);
  • the coaxial locking switch 5 is located on the upper surface of the flange 6 of the pipe body 1 and is rotatable along the upper surface; the coaxial locking switch 5 includes a cross bar 13 and a head 14 disposed at both ends of the cross bar 13, as shown in the figure 8 and FIG. 9, two pairs (four) of locking grooves 15 are provided on the flange 6 at the upper end of the pipe body 1, opposite to each other, and the two ends of the cross bar 13 are located in the pair of locking grooves 15 and are Locking, at this time, the movable shutter 2 is in an open state, and when a certain amount of force directed to the center is applied to the head 14, a certain elastic crossbar 13 is disengaged from the pair of locking grooves 15, maintaining the pair of forces.
  • This embodiment provides a cannula for in vitro purification treatment of a biological sample, which comprises a tube body 1, six activities
  • the baffle 2, the connecting shaft 3, the sealing ring 4, and the coaxial locking switch 5 have the same overall structure as that of the first embodiment, as shown in FIG. 1, wherein the structure of the movable baffle 2 and the polygonal member 11 is as shown in FIG.
  • the movable baffle 2 includes a central portion and an outer portion, the central portion of which has a lower thickness than the outer portion, and when the movable baffles 2 are combined, a disc having a groove in the middle is formed; outside the movable baffle 2
  • the edge end face has a groove which is matched with the size of the sealing ring 4 to allow the sealing ring 4 to be placed thereon to prevent slippage and displacement;
  • the polygonal member 11 is also thinner than the polygonal member 11 of the embodiment 1, and its size is exactly matched with the groove in the middle of the disk formed by the combination of the movable shutters 2, and other structures and embodiments of the sleeve 1 is the same.
  • the embodiment provides a sleeve for in vitro purification treatment of a biological sample, which comprises a tube body 1, six movable baffles 2, a connecting shaft 3, a sealing ring 4, and a coaxial locking switch 5, and the overall structure and implementation thereof Example 1 is the same, as shown in Figure 1, where:
  • the upper end of the tubular body 1 has an outwardly facing flange 6, and the bottom end surface 16 of the tubular body 1 is provided with a first through hole 17 and a plurality of first openings 7 (as shown in FIG. 11), and the bottom end of the side wall is provided a plurality of second openings 9 and a plurality of outwardly extending bumps 8 , wherein the bumps 8 are located above the connecting portion 12 of the adjacent two second openings 9 (as shown in FIG. 6 ); the sealing ring 4 is a rubber ring , sleeved on the outer side of the second opening 9;
  • the movable baffle 2 is disposed at the bottom of the pipe body 1 and has a fan shape.
  • the movable baffle 2 is provided with a plurality of third openings 10, and the outer edge of the movable baffle 2 is in one-to-one correspondence with the second opening 9 and is in contact with the sealing ring 4 ( As shown in FIG. 10, FIG. 3 and FIG. 4; each of the movable baffles 2 has an arcuate notch at the center of the movable baffle 2 (as shown in FIG. 12), and the arcuate notches are combined to form a second through hole 18 ( As shown in FIG.
  • the outer edge end surface of the movable baffle 2 further has a groove matching the size of the sealing ring 4, so that the sealing ring 4 can be placed on the surface to prevent slippage and displacement;
  • the arcuate notch on the movable baffle 2 is not shown in FIG. 10, but this does not mean that the arcuate notch is not on the movable baffle 2;
  • the connecting shaft 3 is located at the center of the pipe body 1, the top end of which is connected to the coaxial locking switch 5, and the bottom end is provided with a polygonal member 11 (in the present embodiment, a regular hexagon), each side of the polygonal member 11 and one
  • the inner edges of the movable baffles 2 are in one-to-one correspondence and contact (as shown in FIGS. 10 and 3);
  • the movable baffle 2 includes a central portion 21 and an outer portion 22 (as shown in FIG. 12), and the central portion 21 has a low thickness.
  • a disc having a groove in the middle (as shown in FIG. 13) is formed, and the size of the polygonal member 11 is exactly the same as that formed by the movable baffle 2
  • the grooves in the middle of the disc match;
  • the bottom portion of the polygonal member 11 has a columnar member 19, and the end of the column member 19 is a spherical locking end 20 having a larger diameter than the first through hole 17 and the second through hole 18; the column member 19 is worn
  • the first through hole 17 is provided on the bottom end surface 16 of the tubular body, and the locking end 20 is stuck outside the first through hole 17 to longitudinally the polygonal member 11 Fixed on the casing tube body, in the absence of longitudinal external force, the polygonal member 11 and the connecting shaft 3 connected thereto can not be longitudinally displaced (as shown in FIG.
  • the curved notch of the movable baffle 2 is composed
  • the second through hole 18 is matched with the first through hole 17 to allow the columnar member 19 at the bottom of the polygonal member 11 to pass through, and the connecting shaft 3 can be easily rotated in the rotating direction;
  • the coaxial locking switch 5 is located on the upper surface of the flange 6 of the pipe body 1 and is rotatable along the upper surface; the coaxial locking switch 5 includes a cross bar 13 and a head 14 disposed at both ends of the cross bar 13, as shown in the figure 8 and FIG. 9, two pairs (four) of locking grooves 15 are provided on the flange 6 at the upper end of the pipe body 1, opposite to each other, and the two ends of the cross bar 13 are located in the pair of locking grooves 15 and are Locking, at this time, the movable shutter 2 is in an open state, and when a certain amount of force directed to the center is applied to the head 14, a certain elastic crossbar 13 is disengaged from the pair of locking grooves 15, maintaining the pair of forces.
  • the embodiment provides a sleeve for in vitro purification treatment of a biological sample, which comprises a tube body 1, six movable baffles 2, a connecting shaft 3, a sealing ring 4, and a coaxial locking switch 5, and the overall structure and implementation thereof Example 1 is the same, as shown in Figure 1, where:
  • the upper end of the tubular body 1 has an outwardly facing flange 6, and the bottom end surface of the tubular body 1 is provided with a plurality of first openings 7 (as shown in FIG. 5), and the bottom end of the side wall is provided with a plurality of second openings 9 and a plurality of outwardly protruding bumps 8 , wherein the bumps 8 are located above the connecting portion 12 of the adjacent two second openings 9 (as shown in FIG. 6 );
  • the sealing ring 4 is a rubber ring which is sleeved on the outer side of the second opening 9;
  • the movable baffle 2 is disposed at the bottom of the pipe body 1, which is a ring-shaped fan, and is provided with a plurality of third openings 10, and the outer edge of the movable baffle 2 is in one-to-one correspondence with the second opening 9 and is in contact with the sealing ring 4. (As shown in FIG. 2, FIG. 15, FIG. 18); and, the inner edge of the movable baffle 2 is provided with a triangular missing U (as shown in FIG. 16), and the triangular missing U can be combined with the apex angle of the polygonal member 11. Matching, when in the open state, the top corner of the polygonal member 11 can fall into the notch on the inner edge of the movable baffle 2 (as shown in Figure 17);
  • the connecting shaft 3 is located at the center of the pipe body 1, the top end of which is connected to the coaxial locking switch 5, and the bottom end is provided with a polygonal member 11 (in the present embodiment, a regular hexagon), each side of the polygonal member 11 and one
  • the inner edges of the movable baffle 2 are in one-to-one correspondence and contact (as shown in FIG. 2 and FIG. 15); for convenience of explanation, the triangular notch on the movable baffle 2 is not shown in FIG. 2, but this does not represent the movable baffle 2 There is no such triangle gap;
  • the coaxial locking switch 5 is located on the upper surface of the flange 6 of the tubular body 1 and is rotatable along the upper surface;
  • the coaxial locking switch 5 includes a cross bar 13 and a head 14 disposed at both ends of the cross bar 13. As shown in FIGS. 8 and 9, two pairs (four) are provided on the flange 6 at the upper end of the pipe body 1.
  • the locking groove 15 is opposite to each other. The two ends of the cross bar 13 are located in a pair of locking grooves 15 and are locked.
  • the movable shutter 2 is in an open state, and when the head 14 is applied with a certain size to the center, When the force is applied, the resilient rod 13 is detached from the pair of locking grooves 15, maintaining the pair of forces and applying a tangential force to the head to cause the crossbar 13 to be transferred into the other pair of locking grooves 15 and to make a horizontal
  • the rod 13 is dropped and locked, and when the crossbar 13 rotates, the connecting shaft 3 and the polygonal member 11 are rotated together, and finally the movable baffle 2 is displaced outward in the radial direction, and is turned from the open state to the closed state.
  • the embodiment provides a sleeve for in vitro purification treatment of a biological sample, which comprises a tube body 1, six movable baffles 2, a connecting shaft 3, a sealing ring 4, and a coaxial locking switch 5, and the overall structure and implementation thereof
  • Example 1 is the same, as shown in Fig. 1, wherein the structure of the movable shutter 2 and the polygonal member 11 is as shown in Fig. 7, and the movable shutter 2 includes a central portion 21 and an outer portion 22 whose central portion 21 has a thickness lower than the outer portion.
  • the triangular notch can be matched with the apex angle of the polygonal member 11, and when in the open state, the apex angle of the polygonal member 11 can fall on the inner edge of the movable baffle 2 The mouth (shown in FIG. 18);
  • the polygonal member 11 is also thinner than the polygonal member 11 of the embodiment 1, and its size is exactly matched with the groove in the middle of the disk formed by the combination of the movable shutters 2, and other structures and embodiments of the sleeve 4 is the same.
  • the present embodiment provides a centrifuge tube for in vitro purification treatment of a biological sample, comprising a common centrifuge tube body and a sleeve provided in Embodiment 1, 2, 3, 4 or 5, the sleeve being located in the body of the centrifuge tube In and with its activities.
  • the movable baffle 2 has two states of closing and opening, as shown in Figures 3 and 4, respectively: wherein: when in the closed state, the six movable baffles 2 are merged with each other to form a circular shape with the polygonal member 11, sealing The ring 4 is also in a contracted state at this time, sleeved on the outside of the second opening 9 of the pipe body 1 and in contact with the outer edge of the movable baffle 2, the first opening 7 at the bottom of the pipe body 1 and the third opening on the movable baffle 2 10 There is no overlapping area, the tube 1 is in a relatively closed state; by rotating the coaxial locking switch 5, the connecting shaft 3 and the polygonal member 11 can be driven to rotate, and when the polygonal member 11 rotates, the side thereof pushes the movable shutter 2 It moves outward in the radial direction, and changes from the closed state to the open state.
  • the movable baffle 2 pushes the sealing ring 4 outward, so that the sealing ring 4 expands and contacts.
  • the gap between the sleeve and the centrifuge tube body is sealed, the first opening 7 at the bottom of the tube body 1 and the third opening 10 on the movable baffle 2 coincide with each other, and the tube body 1 is in an open state.
  • the state allows liquids on the upper and lower sides of the end face of the sleeve to contact each other.
  • the present embodiment provides a method for in vitro purification treatment of a biological sample, which is a method for enriching a single nuclear cell in a blood sample using the centrifuge tube provided in Example 6, and the flow thereof is as shown in FIG.
  • the method includes the following steps:

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Abstract

本发明提供了一种用于生物样本体外纯化处理的套管。该套管包括管体、若干块活动挡板、连接轴、密封圈、同轴锁定开关，其中：管体为筒状，上端具有凸缘，底部端面设有若干个第一开口，侧壁底端设有若干个第二开口以及凸块；密封圈套于第二开口处的外侧；活动挡板设于管体底部，为环扇形，设有若干个第三开口，外缘与第二开口一一对应并与密封圈接触；连接轴位于管体的中心位置，顶端与同轴锁定开关连接，底端设有多边形部件，多边形部件的边与活动挡板的内缘一一对应；同轴锁定开关位于管体的凸缘的上表面并能够转动。

Description

一种用于生物样本体外纯化处理的套管 技术领域
本发明涉及一种用于生物样本体外纯化处理的套管， 属于生物医药技术领域。 背景技术
利用离心法体外富集生物样本中的生物颗粒是生物学研究中应用最为广泛的一项 技术。 许多生物颗粒均可以通过离心法这一技术实现富集， 例如， 动物或植物的细胞、 亚细胞结构、 细菌、 病毒或生物大分子等。 这些在悬液中的生物颗粒在离心作用下的表 现主要取决于生物颗粒的大小和密度， 这可以通过斯托克斯方程 （Stokes equation) 得 到解释。 斯托克斯方程（见下式）描述了球形颗粒在液体介质中受到离心作用时运动速 率的变化规律：
Figure imgf000003_0001
v=
IS n
其中： d代表球形颗粒的直径， p2代表球形颗粒的密度， 表液体介质的密度， n 代表液体介质的粘度， g代表离心力。 通过斯托克斯方程可知， 当颗粒和液体介质的密 度相等时， 颗粒在离心场中的沉降速率为零， 这是利用离心法富集特定生物颗粒的理论 基础。 例如， 利用离心法体外富集人血液样本中的淋巴细胞时， 由于人淋巴细胞的密度 约为 1.077 g/cm3, 可以利用密度同为 1.077 g/cm3的密度介质实现从人血液样本中富集 淋巴细胞的这一过程； 又如， DNA的密度在 1.500-1.800 g/cm3之间， 则应选用可以配制 成该密度范围溶液的氯化铯或硫酸铯等原料作为密度介质配方。
利用离心原理进行生物样本体外纯化处理， 即从生物样本中富集生物颗粒的步骤是 基本相似的。 例如， 利用密度介质进行血液中人淋巴细胞的体外富集时， 其一般操作步 骤包括： （1 )用一定比例的平衡盐溶液或生理盐水对血液样本进行预稀释； （2)将稀释 后的血液样本小心地沿离心容器壁加入预装有密度介质的离心容器中，保持样本与介质 的界面清晰，不发生混合，这一过程是保证富集效果的关键步骤，常被称为"铺血"； (3 ) 在一定温度和一定离心条件下进行离心，例如 20°C时，离心力为 400g的条件下离心 30 分钟； （4) 小心从离心机中取出装有样本的离心容器， 利用巴斯德吸管或移液管等装置 吸取富集在样本和介质层交界面处的 "白膜层"； ( 5 ) 用一定比例的平衡盐溶液、 生理 盐水或细胞培养基洗涤得到的细胞产品， 除去血小板、 细胞碎片与部分死细胞， 以及残 余的密度介质； （6) 将得到的细胞悬液用于细胞培养等后续实验。
为实现良好的富集效果， 上述操作步骤对于操作者的技术要求是十分高的。 例如， 在上述步骤 （2) 的操作中， 操作者必须将稀释后的血液样本非常小心地缓缓铺在密度 介质之上， 保持样本与介质的界面清晰， 不发生混合。 这是因为， 如果血液样本与密度 介质发生混合， 最终富集在样本和介质层之间的 "白膜层 "将变得非常松散， 且含有大 量红细胞污染， 这不仅会给步骤 （4) 中的收集操作带来视觉判断上的障碍， 还会对目 的细胞的收率和纯度产生严重的不良影响。 因此， "铺血"这一操作的特点是既费时、 又费力， 对于操作者的技术要求高， 不利于实现规模化处理， 特别是用于临床治疗用途 时， 大量血液样本的规模化处理是一大技术难题。 又例如， 在上述歩骤 （4) 的操作中， 操作者首先需要对 "白膜层"的位置进行准确判断， 然后利用巴斯德吸管或移液管等装 置伸入液面以下吸取富集在这一区域的目的细胞， 这不仅费时费力， 且随着目的细胞不 断地被吸出， 白膜层的厚度逐渐缩小， 外观和位置也越来越不明显， 这对于操作者何时 应停止吸取操作的判断再次提出了较高的要求， 如果操作者对细胞的余量出现判断上的 失误， 提前停止了吸取操作， 则目的细胞的收率将明显降低。
基于上述操作中的困难和问题， 一些解决方案被相继提出。 例如， 一些国外企业开 发了专门用于从血液样本中纯化淋巴细胞的离心管， 有代表性的包括德国 Greiner bio-one公司开发的产品 Leucosep®离心管，该离心管的特点是在管内略下部安装固定了 一层多孔滤网屏障， 将离心管分为了上下两个隔室。 由于屏障的孔径比较小， 血液样本 无法立刻透过屏障， 这样可以将血液样本直接倾倒在下隔室预装有密度介质的离心管 中， 而不必担心血液样本与密度介质发生混合。 在后续的离心操作中， 由于血细胞的半 径远小于屏障的孔径， 因此血细胞可以自由通过滤网屏障并与介质层接触， 而不影响分 层效果。 这样的装置较好地解决了上述步骤 （2) 中所述的 "铺血"操作中的困难， 大 大缩短了操作时间， 提高了工作效率和效果。 然而， 这种具有多孔滤网屏障结构的离心 管的缺陷也是十分明显的， 具体而言包括如下几点： （a) 由于屏障的孔径比较小， 不仅 血液样本无法立刻透过屏障， 密度介质同样也无法立刻透过屏障， 因此适用于这种装置 的标准操作是先向上隔室加入适量的密度介质， 比如 Ficoll-PaqueTM Plus, 然后通过数 分钟的离心， 使密度介质透过屏障全部进入下隔室， 方可进行后续的 "铺血"操作， 这 再次给血液样本的分离操作引入了额外的麻烦。 此外， 所加入的密度介质的量需要得到 非常准确的控制， 其应略低于屏障的高度， 如果加入量过少， 对纯化效果将产生不利影 响； 如果加入量过多， 则还需要回收超过屏障高度的密度介质， 这同样为分离操作带来 了不便。 （b)具有多孔滤网屏障结构的离心管的生产工艺比较复杂， 目前仅国外几家企 业能够完成生产加工， 而国内缺乏成熟的技术和相应的产品。 这主要因为， 在光滑的离 心管壁上安装固定一层多孔滤网屏障结构对粘合技术提出了十分高的要求。粘合不仅需 要足够的机械强度以保证运输和离心操作中屏障不会脱落， 更重要的是， 粘合剂的选择 需要考虑不溶于水、 对细胞无毒无害等生物安全性因素。 粘合剂的问题给这类产品潜在 的临床应用转化带来了巨大阻碍。 （C )基于技术工艺的复杂性， 该产品造价昂贵， 每支 具有多孔滤网屏障结构的离心管是同样容量离心管价格的约几十倍至一百倍，这大大限 制了该类产品的广泛使用。
为解决上述缺陷 （a) 中所述的问题， 美国 Sigma 公司开发了产品 ACCUSPIN™ System-Histopaque®-1077, 它是一种预装有适量密度介质的具有多孔滤网屏障结构的离 心管。 预装密度介质的解决方案的确很好解决了上述缺陷 （a) 中所述的问题， 然而又 带来了如下所述的缺陷： （d ) 此类产品的储存方式必须针对密度介质而单独设定。 ACCUSPIN™ System-Histopaque®-1077 的说明书中明确指出， 这种预装有适量密度介 质的具有多孔滤网屏障结构的离心管必须在 2-8 °C、 避光条件下保存， 由于离心管所占 用的体积较大， 这种特殊的保存方式为这类产品的应用带来了很大不便。 （e)在水性密 度介质长期的浸泡下， 滤网屏障的粘合剂效果及溶出效果是否还能满足使用要求缺乏充 分的试验数据支持。 综上， 预装有适量密度介质的具有多孔滤网屏障结构的离心管不仅 无法解决上述缺陷中 （b)、 ( c) 所述的问题， 还额外引入了缺陷 （d) 和 （e ) 中所述的 保存、 产品安全性和有效性的问题。
中国专利 200610063125.4 提供了一种在管底设置了疏松多孔材料的淋巴细胞分离 管， 该疏松多孔材料可以将密度介质吸附成为半固态， 在进行血液样本的纯化时， 可以 将血液样本直接倒入这种分离管， 而不必担心弄混血液与分离液二者的界面， 很好解决 了 "铺血"操作费时费力的问题。 并且， 由于所述的疏松多孔材料与管体之间通过摩擦 力即可实现固定， 因而这种技术不需要使用粘合剂， 工艺难度大大降低， 因而克服了上 述缺陷中 （b ) 和 （c) 中所述的问题。 然而， 这种在管底设置了疏松多孔材料的淋巴细 胞分离管仍然面临密度介质加入过程的耗时问题， 由于疏松多孔材料可以阻止血液立刻 透过， 密度介质的吸附和渗透也需要一定时间， 需要通过 "滴加"才能实现； 并且， 由 于疏松多孔材料本身并不是透明的， 密度介质的加入量也较难实现准确控制， 这进而对 于血液样本分离效果的控制和结果的可重复性带来了很大困难。上述专利还提供了另一 种产品形式， 即预先将淋巴细胞分离液（密度介质）加入管体的疏松多孔材料中， 这虽 然可以实现 "即开即用"和密度介质加入量的准确控制， 然而， 疏松多孔材料的材质同 样面临着上述缺陷 （d) 和 （e) 中所述的保存、 产品安全性和有效性的问题。 即， 预加 入的密度介质将长期浸泡在疏松多孔材料中， 疏松多孔材料（例如上述专利中记载的特 种海绵）是否在这种浸泡条件下会溶出对于血细胞有害的成分， 这需要通过大量试验进 一步证明。 发明内容
为解决上述技术问题，本发明的目的在于提供一种用于生物样本体外纯化处理的套 管， 该套管可以装入离心容器之中， 以用于生物样本的体外纯化处理， 该套管具有方便 实用、 生产工艺简单、 成本低以及安全无毒的特点， 并特别适合用于生物样本的规模化 处理， 使操作过程得到了极大程度的简化。
本发明的目的还在于提供一种包括上述套管的离心容器。
本发明的目的还在于提供一种利用上述离心容器进行的生物样本体外纯化处理的 方法。
为达到上述目的， 本发明首先提供了一种用于生物样本体外纯化处理的套管， 其包 括管体、 若干块活动挡板、 连接轴、 密封圈、 同轴锁定开关， 其中：
所述管体为筒状 （优选为圆筒状）， 其上端具有外向的凸缘， 其底部端面上设有若 干个第一开口， 其侧壁的底端设有若干个第二开口以及若干个外向的凸块， 其中， 所述 凸块位于相邻两个所述第二开口的连接部的上方；
所述密封圈套于所述第二幵口处的外侧；
所述活动挡板设于所述管体的底部， 所述活动挡板上设有若干个第三开口， 所述活 动挡板的外缘与所述第二开口一一对应并与所述密封圈接触；
所述连接轴位于所述管体的中心位置， 其顶端与所述同轴锁定开关连接， 底端设有 一多边形部件， 该多边形部件的每一条边与一个所述活动挡板的内缘一一对应并接触； 所述同轴锁定开关位于所述管体的凸缘的上表面上并且能够沿该上表面转动； 当所述活动挡板处于打开状态时， 所述第一开口和第三开口之间至少部分重合； 当 所述活动挡板处于闭合状态时， 所述第一开口和第三开口之间不存在重合的部分， 所述 第二开口的形状和尺寸与所述活动挡板的外缘相匹配。
本发明提供的上述套管是用于装入离心容器（例如普通离心管、 离心瓶） 中的， 以 便实现对生物样本中的生物颗粒的富集。 为便于将套管放入离心容器中或者将其取出， 该套管管体的外径略小于离心容器的内径，二者活动套配，而该套管的长度（或称高度） 可以根据需要结合离心容器的尺寸进行确定。该套管顶部的凸缘的尺寸以能够使套管挂 在离心容器上进行固定， 并且不影响离心容器盖的使用为宜， 优选等于或略小于离心容 器的螺纹外径。位于套管管体下部的凸块的横向尺寸（外径或者相对于管体外壁的高度) 应使该凸块能够与离心容器内壁较为紧密地接触、又要使套管能够顺利放入离心容器中 和取出， 优选地， 凸块外缘所组成的圆形的直径与离心容器的内径相同或者略小于其内 径。另外， 该凸块的底面应与密封圈相互接触， 以便能够压住密封圈避免其上翘， 同时， 在活动挡板处于打开状态时， 该凸块也可以压住活动挡板的外沿避免其上翘， 还可以与 橡胶圈一起将活动挡板打开之后产生的部分缝隙密封住。
在上述套管中， 活动挡板安装在管体的底部， 并且可以沿半径方向在一定范围内自 由运动。 活动挡板的外缘为弧形、 连接在一起可以形成一个圆。 每一块活动挡板上都设 有一定数量的第三开口， 以便生物样本中的生物颗粒能够通过。 活动挡板具有打开和闭 合两种状态， 当活动挡板处于闭合状态时， 全部的活动挡板组成一个封闭的环状， 外缘 连在一起形成一个封闭的圆， 并且， 中心的多边形空洞或多边形凹槽正好由连接轴底端 的多边形部件填充， 由此， 活动挡板与连接轴底端的多边形部件形成封闭的圆形或者圆 盘。根据本发明的具体实施方案，优选地，本发明提供的套管包括 3-12个（优选 3-6个） 活动挡板， 第二开口的数量、 凸块的数量、 多边形部件的边数与活动挡板的数量相同。 对应于 3-12个活动挡板， 连接轴底部的多边形部件可以为正三角形-正十二边形， 对应 于 3-6个活动挡板， 连接轴底部的多边形部件可以为正三角形 -正六边形。
根据本发明的具体实施方案， 优选地， 活动挡板为环扇形， 并且， 活动挡板的厚度 与多边形部件的厚度相同， 所有活动挡板与多边形部件组成一个完整的圆盘形； 或者活 动挡板为扇形， 挡板的中心部分的厚度低于外侧部分的厚度， 并且， 活动挡板的中心部 分的厚度与多边形部件的厚度之和等于或大于活动挡板外侧部分的厚度，所有活动挡板 与多边形部件组成一个完整的圆盘形。 当活动挡板为环扇形时， 其内缘可以为直线型， 所有活动挡板组成一个中间具有多边形孔洞的圆盘， 该多边形孔洞与连接轴底端的多边 形部件的形状相同； 当活动挡板为扇形时， 其中心部分较之外半部分薄一些， 所有活动 挡板的中心部分结合在一起形成一个中间有凹槽的圆盘， 该凹槽的边优选为直线型， 其 形状与多边形部件能够相互配合， 多边形部件也略薄一些以便与活动挡板的厚度匹配， 通过将活动挡板的中间部分压在多边形部件的下面，可以使活动挡板较好地设置在套管 底部， 不会发生翘起， 以免影响密封效果。
根据本发明的具体实施方案， 优选地， 活动挡板的外缘侧面具有一与密封圈相配合 的凹槽。 密封圈可以套在该凹槽内， 保持牢固性， 以免发生滑脱。
根据本发明的具体实施方案， 优选地， 所述管体的底部端面上设有第一通孔， 所述 活动挡板为扇形， 其圆心处具有一弧形缺口， 所述挡板的中心部分的厚度低于外侧部分 的厚度，所述活动挡板的中心部分的厚度与所述多边形部件的厚度之和等于或大于所述 活动挡板外侧部分的厚度， 所有活动挡板与所述多边形部件组成一个完整的圆盘形， 所 有所述活动挡板的弧形缺口组合在一起构成一个与所述第一通孔对应的第二通孔； 所述多边形部件的底部中心位置具有一柱状部件， 该柱状部件穿过所述第二通孔和 所述第一通孔， 该柱状部件的末端为一卡止端， 该卡止端将所述连接轴和多边形部件纵 向固定。
在上述套管中， 第一通孔用于使多边形部件底部的柱状部件穿过， 该柱状部件末端 的卡止端位于底部端面以下， 其可以将多边形部件以及连接轴进行纵向固定， 防止其产 生不需要的纵向位移， 这可以避免活动挡板在转动时上翘， 以及连接轴在转动时发生纵 向位移， 进而造成活动挡板的非预期位移。
在上述套管中， 设于多边形部件底部的柱状部件优选是设置在多边形部件底部的中 心位置， 沿着连接轴中心线的延长线方向。 该柱状部件的中间部分较细， 末端的卡止端 可以是任何实现卡止功能的立体形状， 优选为球形、 长方体形、 多面体形等， 只要其尺 寸能够大于第一通孔从而卡在第一通孔处将多边形部件和连接轴纵向固定即可。 优选 地， 所述卡止端的水平尺寸大于所述第一通孔的直径。 在套管的管体底部端面上开设的 第一通孔用于使多边形部件底部的柱状部件穿过， 该柱状部件末端的卡止端位于底部端 面以下， 其可以将多边形部件以及连接轴进行纵向固定在套管管体上， 防止其产生不需 要的纵向位移， 这可以避免活动挡板在转动时上翘， 以及避免连接轴在转动时发生纵向 位移， 避免进而造成的活动挡板的非预期位移。
根据本发明的具体实施方案， 优选地， 所述活动挡板的内缘设有与所述多边形部件 的角相匹配的缺口。 在上述套管中， 活动挡板上的缺口是设置在与多边形部件的边相接 触的内缘上。 当活动挡板为环扇形时， 上述缺 U是设置在环扇形的直线型内缘上； 当活 动挡板为扇形时， 上述缺口是设置在活动挡板中心部分与外侧部分的交界线上， 即所有 活动挡板组合在一起时所形成的凹槽的边缘上。 上述的凹槽朝向中心， 其形状可以是任 何能够使多边形部件的一角嵌入其中起到转动锁定效果的形状， 例如半圆形、 三角形， 优选为三角形。
在套管的管体底部端面上开设的第一幵口是用于使生物样本中的生物颗粒（例如血 细胞）在离心场中可以自由通过并与装在离心容器中的密度介质进行接触。 该第一开口 的形状和大小可以根据需要进行选择， 但要与活动挡板上的第三开口相适应， 以便在打 开状态时使生物样本能够通过第一开口与第三开口的重合部分与密度介质接触。根据本 发明的具体实施方案， 优选地， 当活动挡板处于打开状态时， 第一开口和第三开口之间 至少部分重合（优选为完全重合， 此种情况下， 对应位置的第一开口和第三开口可以具 有相同的形状）； 当活动挡板处于闭合状态时， 第一开口和第三开口之间不存在重合的 部分， 即互相封闭。
在套管的侧壁上设置的第二开口可以根据需要进行设置，其数量应与活动挡板的数 量一致， 并且， 该第二开口应使活动挡板能够在打开状态时自由出入并满足一定的封闭 需要， 即第二开口的尺寸及形状应与活动挡板的外缘尺寸及形状相匹配。 在管体侧壁上 设置的凸块的数量也与活动挡板的数量相同。 上述第二开口沿着套管的外壁设置， 其可 以为矩形 （或称曲面的矩形）， 即上边和下边分别与套管的底部端面平行， 左边和右边 分别与套管的底部端面垂直。
在本发明提供的套管中， 密封圈套设于第二开口的外侧， 以便在使用过程中对套管 和离心容器之间的缝隙进行密封。 根据本发明的具体实施方案， 优选地， 密封圈的粗细 与第二开口的高度相同。 该密封圈可以是任何能够实现密封的圈， 优选为有弹性的密封 圈， 例如橡胶圈。 该密封圈的内径可以与管体的外径相同， 以便于套设。 该密封圈的内 缘与活动挡板的外缘接触，各个活动挡板的外缘连在一起所组成的圆的直径应与密封圈 的内径相同。
在本发明提供的套管中， 当活动挡板处于封闭状态时， 其处于 "收缩"状态， 密封 圈也处于 "收缩"状态 （没有弹性形变的状态）， 其外径略小于离心容器的内径， 套管 管体与离心容器之间的缝隙不会被密封， 套管管体可以自由从离心容器中取出或放入， 此时活动挡板上的第三开口与管体底部的第一开口彼此间通过相差相互遮挡， 管体处于 相对闭合的状态， 生物样本不能通过； 而当活动挡板处于打开状态时， 其处于 "扩张" 状态， 活动挡板上的第三开 U与管体底部的第一开 U彼此至少部分重合， 生物样本可以 自由通过， 同时， 密封圈也随着活动挡板的扩张， 被活动挡板的边缘沿半径方向向外推 挤， 变为 "扩张"状态并与离心容器外壁紧密接触， 并且连同管体下部的凸块一起将套 管管体与离心容器内壁之间的缝隙密封（此时密封圈的外径达到离心容器内径的尺寸）， 生物样本不能通过， 即使在离心场中也无法通过该缝隙。
在上述套管中， 连接轴底端的多边形部件与活动挡板的靠中心侧紧密接触， 活动挡 板的状态切换 （打开和闭合）是通过多边形部件、 连接轴及同轴锁定开关的转动同步实 现的。 相应地， 多边形部件也可以有打开和闭合两种使用状态（此处的打开和闭合的描 述是为了与活动挡板的状态保持一致而定义的， 并不意味着多边形部件本身的形状要被 打开或者闭合）， 多边形部件处于闭合状态时， 多边形部件的边缘与活动挡板的靠中心 侧边缘平行接触， 活动挡板处于闭合状态， 密封圈收缩， 管体处于相对闭合状态； 多边 形部件处于打开状态时， 多边形部件的顶点与活动挡板的靠中心侧边缘接触， 活动挡板 沿半径方向向外产生一定距离的位移， 活动挡板上的第三开口与管体底部的第一开口彼 此至少部分重合， 由闭合状态转到打开状态。
根据本发明的具体实施方案，对于活动挡板的内缘设有与所述多边形部件的角相匹 配的缺口的套管， 当多边形部件处于闭合状态时， 多边形部件的边缘与活动挡板的靠中 心侧边缘平行接触， 活动挡板处于闭合状态， 密封圈收缩， 管体处于相对闭合状态； 当 转动同轴锁定开关时， 连接轴和多边形部件也会转动， 在此过程中， 多边形部件的顶角 会推动活动挡板向外产生位移， 直到同轴锁定开关转到预定位置， 多边形部件到达打开 状态； 多边形部件处于打开状态时， 多边形部件的顶角嵌入活动挡板的靠中心侧边缘上 的缺口中， 活动挡板沿半径方向向外产生一定距离的位移， 活动挡板上的第三开口与管 体底部的第一开口彼此至少部分重合， 由闭合状态转到打开状态。 此时， 多边形部件的 每个顶角都嵌入到相应的缺口中， 不施加转动方向外力的情况下， 多边形部件在搬运或 离心状态下将能够在转动方向上得到锁定。 该缺口 （与同轴锁定开关一起）保证了活动 挡板在使用过程中不会发生非预期的移动， 从而保证了套管使用的良好稳定性。
在上述套管中， 连接轴位于管体的中心、 与管体的中心轴重合， 其顶端与同轴锁定 开关连接。 同轴锁定开关可以通过旋转带动连接轴转动， 从而带动改变多边形部件的位 置状态， 进而实现活动挡板在闭合和打开状态之间的切换。 在活动挡板处于闭合和打开 的两种位置状态下时， 同轴锁定开关均可以通过一定的锁定结构实现在管体上端环形凸 缘的位置锁定， 避免活动挡板在离心场中或搬运状态下发生非预期的位移。 上述锁定结 构可以是任何能够实现上述锁定的装置， 优选地， 套管的凸缘上设有至少两对 （四个） 锁定槽， 该锁定槽两两相对， 同轴锁定开关包括一横杆， 该横杆中间与连接轴顶端连接 并能够带动连接轴转动，其两端分别位于一对锁定槽之中并能够在不同的锁定槽之间转 换。 例如， 在套管的凸缘上设置四个锁定槽 （凹槽）， 使其两两相对， 同轴锁定结构的 横杆中间与连接轴顶端连接并能够带动连接轴转动， 其两端分别位于一对锁定槽之中， 在外力作用下该横杆可以转动到下一对锁定槽中，通过转动可以实现活动挡板在打开和 关闭状态之间的转换。 横杆的形状只要适合锁定槽形状即可， 横杆的尺寸与锁定槽的尺 寸相当， 使横杆在落入锁定槽时有一定的阻力， 保证横杆在纵向方向上通过阻力得到固 定； 该横杆的两端可以包括两个向上的杆头， 以便于施加外力转动横杆及对横杆两端施 加指向中心的两个力， 使横杆两头翘起脱离一对锁定槽， 并保持横杆在两头翘起状态时 可以沿套管的凸缘滑动到另一对锁定槽中； 横杆在锁定槽内时， 其在转动方向上会被锁 定， 以实现活动挡板的位置和状态锁定、 保证活动挡板不会任意的打开； 同时， 由于横 杆在纵向方向上通过与锁定槽之间的阻力得到固定， 当多边形部件底部的柱状部件末端 具有卡止端时， 该卡止端卡在管体底部端面上的第一通孔的外侧， 因此， 整个连接轴连 同多边形部件不会上下活动， 在纵向方向上也实现了锁定； 同时， 通过增加卡止端也可 以避免活动挡板在转动时发生上翘。当指向中心的一对外力作用于横杆两端并达到一定 大小时， 才能够使横杆从锁定槽中暂时脱离出来并转到另一对锁定槽中被锁定， 该横杆 优选具有一定的弹性， 以便于被转进转出； 两对锁定槽之间的距离可以根据活动挡板的 数量进行确定， 只要能够实现活动挡板的打开和闭合状态之间的转换以及锁定即可。
本发明所提供的套管所采用的材料可以是任何满足相关卫生标准的医用材料，例如 该套管的材料可以是用于制备临床注射用的注射器的塑料材料，密封所采用的橡胶圈的 橡胶材料可以是符合卫生标准的医用橡胶，例如用于制作临床注射用的注射器推杆前段 的橡胶部件的橡胶材料， 例如医用硅橡胶等。
本发明还提供了一种用于生物样本体外纯化处理的离心容器，其包括上述用于生物 样本体外纯化处理的套管和离心容器本体， 其中， 套管位于离心容器本体之中并且二者 活动套配。
根据本发明的具体实施方案， 优选地， 上述离心容器可以为离心管或离心瓶等。 本发明还提供了一种用于生物样本体外纯化处理的方法，其是采用上述离心容器对 生物样本中的生物颗粒进行富集， 其包括以下步骤：
将套管装配在离心容器中， 并打开套管的活动挡板；
将密度介质装入离心容器本体中， 关闭套管的活动挡板；
将生物样本注入套管中；
打开套管的活动挡板， 使生物样本与密度介质接触， 进行离心处理， 离心处理之后 在套管的活动挡板上方形成生物颗粒富集层；
关闭套管的活动挡板， 将套管从离心容器本体中取出， 将生物颗粒富集层转移， 完 成生物样本中生物颗粒的富集。
通过上述方法分离得到的生物颗粒富集层可以通过适当的处理之后用于不同用途， 具体的处理可以根据常规方式进行。 在上述方法中， 装入的密度介质的量可以根据需要确定， 优选地， 装入离心容器本 体中的密度介质的上表面与套管的底端面接触或略高于底端面。
本发明所提供的上述方法可以用于现有生物样本的富集， 优选地， 上述生物样本为 血液或组织样本等， 生物样本中的生物颗粒为淋巴细胞、 单核细胞、 造血干细胞、 间充 质干细胞等。
本发明所提供的套管是针对密度梯度离心法从血液或组织样本等生物样本中富集 生物颗粒时所存在的困难和问题而提出的， 可以解决 "铺血"操作和 "吸取白膜层"操 作费时费力且不利于规模化处理生物样本的问题； 也能够解决背景技术中所提供的现有 产品或技术所存在的 （a) - ( e) 中所述的缺陷。 本发明所提供的套管是一种方便实用、 生产成本低廉、 生产工艺简单且安全无毒的用于生物样本体外纯化处理的装置。 附图说明
图 1为实施例 1提供的套管的整体结构示意图；
图 2为实施例 1提供的套管的活动挡板与连接轴的爆炸示意图；
图 3为实施例 1提供的套管的活动挡板的闭合状态示意图；
图 4为实施例 1提供的套管的活动挡板的打开状态示意图；
图 5为实施例 1提供的套管底部端面的第一开口示意图；
图 6为实施例 1提供的套管侧面的第二幵口和凸块示意图；
图 7为实施例 2中的活动挡板与多边形部件的结构示意图；
图 8为实施例 1中同轴锁定开关的设置方式示意图；
图 9为图 8的 A-A向截面图；
图 10为实施例 3提供的套管的活动挡板与连接轴的爆炸示意图；
图 11为实施例 3提供的套管底部端面的第一开口和第一通孔示意图；
图 12为实施例 3提供的单个活动挡板的示意图；
图 13为实施例 3提供的活动挡板的弧形缺口组成的第二通孔的示意图； 图 14为实施例 3中的活动挡板与多边形部件的结构示意图；
图 15为实施例 4提供的套管的活动挡板的闭合状态示意图；
图 16为实施例 4提供的带有缺口的活动挡板的示意图；
图 17为实施例 5提供的带有缺口的活动挡板的示意图；
图 18为实施例 4提供的套管的活动挡板的打开状态示意图；
图 19为实施例 7提供的用于生物样本体外纯化处理的方法的流程示意图。 主要组件符号说明：
管体 1 活动挡板 2 连接轴 3 密封圈 4 同轴锁定开关 5 凸缘 6 第一开口 7 凸块 8 第二开口 9 第三开口 10 多边形部件 11 连接部 12 横杆 13 杆头 14 锁定槽 15 底部端面 16 第一通孔 17 第二通孔 18 柱状部件 19 卡止端 20 中心部分 21 外侧部分 22 具体实施方式
为了对本发明的技术特征、 目的和有益效果有更加清楚的理解， 现对本发明的技术 方案进行以下详细说明， 但不能理解为对本发明的可实施范围的限定。
实施例 1
本实施例提供了一种用于生物样本体外纯化处理的套管， 其包括管体 1、 六块活动 挡板 2、 连接轴 3、 密封圈 4、 同轴锁定开关 5， 如图 1所示， 其中：
管体 1的上端具有外向的凸缘 6， 管体 1的底部端面上设有若干个第一开口 7 (如 图 5所示）， 其侧壁的底端设有若干个第二开口 9以及若干个外向的凸块 8， 其中， 凸块 8位于相邻两个第二开口 9的连接部 12的上方 （如图 6所示）；
密封圈 4为一橡胶圈， 套于第二开口 9处的外侧；
活动挡板 2设于管体 1的底部， 其为环扇形， 其上设有若干个第三开口 10， 该活动 挡板 2的外缘与第二幵口 9一一对应并与密封圈 4接触（如图 1、图 2、图 3、图 4所示）； 连接轴 3位于管体 1的中心位置， 其顶端与同轴锁定开关 5连接， 底端设有一多边 形部件 11 (在本实施例中为正六边形）， 该多边形部件 11的每一条边与一个活动挡板 2 的内缘一一对应并接触 （如图 2、 图 3所示）；
同轴锁定开关 5位于管体 1的凸缘 6的上表面上并且能够沿该上表面转动； 该同轴锁定开关 5包括一横杆 13和设于横杆 13两端的杆头 14，如图 8和图 9所示， 在管体 1上端的凸缘 6上设有两对 （四个） 锁定槽 15， 两两相对， 横杆 13的两端位于 其中一对锁定槽 15之中并被锁定， 此时活动挡板 2处于打开状态， 当对杆头 14施加一 定大小的指向中心的一对力时， 具有一定弹性的横杆 13从这一对锁定槽 15中脱离， 保 持这对力并对杆头施加切向外力使横杆 13转到另一对锁定槽 15之中并使横杆 13落入 而被锁定， 横杆 13转动时会带动连接轴 3、 多边形部件 11一起转动， 最终使活动挡板 2沿半径方向向外产生一定位移， 其由打开状态转到闭合状态。
实施例 2
本实施例提供了一种用于生物样本体外纯化处理的套管， 其包括管体 1、 六块活动 挡板 2、 连接轴 3、 密封圈 4、 同轴锁定开关 5， 其整体结构与实施例 1相同， 如图 1所 示， 其中， 活动挡板 2和多边形部件 11的结构如图 7所示， 活动挡板 2包括中心部分 和外侧部分， 其中心部分的厚度低于外侧部分， 当活动挡板 2组合在一起时， 会形成一 个中间有凹槽的圆盘； 该活动挡板 2的外缘端面具有一凹槽， 该凹槽与密封圈 4的尺寸 匹配， 可以使密封圈 4套在上面， 防止滑脱和移位；
而多边形部件 11也相对于实施例 1中的多边形部件 11要薄一些， 其尺寸正好与活 动挡板 2组合在一起形成的圆盘中间的凹槽相匹配，该套管的其他结构与实施例 1相同。
实施例 3
本实施例提供了一种用于生物样本体外纯化处理的套管， 其包括管体 1、 六块活动 挡板 2、 连接轴 3、 密封圈 4、 同轴锁定开关 5， 其整体结构与实施例 1相同， 如图 1所 示， 其中：
管体 1的上端具有外向的凸缘 6， 管体 1的底部端面 16上设有第一通孔 17和若干 个第一开口 7 (如图 11所示）， 其侧壁的底端设有若干个第二开口 9以及若干个外向的 凸块 8， 其中， 凸块 8位于相邻两个第二开口 9的连接部 12的上方 （如图 6所示）； 密封圈 4为一橡胶圈， 套于第二开口 9处的外侧；
活动挡板 2设于管体 1的底部， 其为扇形， 其上设有若干个第三开口 10， 该活动挡 板 2的外缘与第二开口 9一一对应并与密封圈 4接触 （如图 10、 图 3、 图 4所示）； 每 一块活动挡板 2的圆心处均具有一弧形缺口 （如图 12所示）， 这些弧形缺口组合在一起 形成第二通孔 18 (如图 13所示）； 活动挡板 2的外缘端面还具有一凹槽， 该凹槽与密封 圈 4的尺寸匹配， 可以使密封圈 4套在上面， 防止滑脱和移位； 为便于说明， 图 10中 未显示活动挡板 2上的弧形缺口， 但这不代表活动挡板 2上没有该弧形缺口；
连接轴 3位于管体 1的中心位置， 其顶端与同轴锁定开关 5连接， 底端设有一多边 形部件 11 (在本实施例中为正六边形）， 该多边形部件 11的每一条边与一个活动挡板 2 的内缘一一对应并接触（如图 10、 图 3所示）； 活动挡板 2包括中心部分 21和外侧部分 22 (如图 12所示）， 其中心部分 21的厚度低于外侧部分 22， 当活动挡板 2组合在一起 时， 会形成一个中间有凹槽的圆盘 （如图 13所示）， 多边形部件 11的尺寸正好与活动 挡板 2组合在一起形成的圆盘中间的凹槽相匹配；
多边形部件 11的底部中心位置处具有一柱状部件 19，该柱状部件 19的末端为一圆 球形的卡止端 20， 其直径大于第一通孔 17和第二通孔 18; 该柱状部件 19穿设于管体 底部端面 16上的第一通孔 17，其卡止端 20卡在第一通孔 17外侧将多边形部件 11纵向 固定在套管管体上， 在没有纵向外力的情况下， 该多边形部件 11 以及与之连接的连接 轴 3均无法发生纵向位移 （如图 14所示）； 活动挡板 2的弧形缺口组成的第二通孔 18 与第一通孔 17相匹配， 能够允许多边形部件 11底部的柱状部件 19穿过， 并能够实现 连接轴 3在转动方向较容易地旋转；
同轴锁定开关 5位于管体 1的凸缘 6的上表面上并且能够沿该上表面转动； 该同轴锁定开关 5包括一横杆 13和设于横杆 13两端的杆头 14，如图 8和图 9所示， 在管体 1上端的凸缘 6上设有两对 （四个） 锁定槽 15， 两两相对， 横杆 13的两端位于 其中一对锁定槽 15之中并被锁定， 此时活动挡板 2处于打开状态， 当对杆头 14施加一 定大小的指向中心的一对力时， 具有一定弹性的横杆 13从这一对锁定槽 15中脱离， 保 持这对力并对杆头施加切向外力使横杆 13转到另一对锁定槽 15之中并使横杆 13落入 而被锁定， 横杆 13转动时会带动连接轴 3、 多边形部件 11一起转动， 最终使活动挡板 2沿半径方向向外产生一定位移， 其由打开状态转到闭合状态。
实施例 4
本实施例提供了一种用于生物样本体外纯化处理的套管， 其包括管体 1、 六块活动 挡板 2、 连接轴 3、 密封圈 4、 同轴锁定开关 5， 其整体结构与实施例 1相同， 如图 1所 示， 其中：
管体 1的上端具有外向的凸缘 6， 管体 1的底部端面上设有若干个第一开口 7 (如 图 5所示）， 其侧壁的底端设有若干个第二开口 9以及若干个外向的凸块 8， 其中， 凸块 8位于相邻两个第二开口 9的连接部 12的上方 （如图 6所示）；
密封圈 4为一橡胶圈， 套于第二开口 9处的外侧；
活动挡板 2设于管体 1的底部， 其为环扇形， 其上设有若干个第三开口 10， 该活动 挡板 2的外缘与第二开口 9一一对应并与密封圈 4接触 （如图 2、 图 15、 图 18所示）； 并且， 该活动挡板 2的内缘上设有三角形缺 U (如图 16所示）， 该三角形缺 U能够与多 边形部件 11的顶角相匹配， 当处于打开状态时， 多边形部件 11的顶角能够落入活动挡 板 2内缘上的缺口中 （如图 17所示）；
连接轴 3位于管体 1的中心位置， 其顶端与同轴锁定开关 5连接， 底端设有一多边 形部件 11 (在本实施例中为正六边形）， 该多边形部件 11的每一条边与一个活动挡板 2 的内缘一一对应并接触 （如图 2、 图 15所示）； 为便于说明， 图 2中未显示活动挡板 2 上的三角形缺口， 但这不代表活动挡板 2上没有该三角形缺口；
同轴锁定开关 5位于管体 1的凸缘 6的上表面上并且能够沿该上表面转动； 该同轴锁定开关 5包括一横杆 13和设于横杆 13两端的杆头 14，如图 8和图 9所示， 在管体 1上端的凸缘 6上设有两对 （四个） 锁定槽 15， 两两相对， 横杆 13的两端位于 其中一对锁定槽 15之中并被锁定， 此时活动挡板 2处于打开状态， 当对杆头 14施加一 定大小的指向中心的一对力时， 具有一定弹性的横杆 13从这一对锁定槽 15中脱离， 保 持这对力并对杆头施加切向外力使横杆 13转到另一对锁定槽 15之中并使横杆 13落入 而被锁定， 横杆 13转动时会带动连接轴 3、 多边形部件 11一起转动， 最终使活动挡板 2沿半径方向向外产生一定位移， 其由打开状态转到闭合状态。
实施例 5
本实施例提供了一种用于生物样本体外纯化处理的套管， 其包括管体 1、 六块活动 挡板 2、 连接轴 3、 密封圈 4、 同轴锁定开关 5， 其整体结构与实施例 1相同， 如图 1所 示， 其中， 活动挡板 2和多边形部件 11的结构如图 7所示， 活动挡板 2包括中心部分 21和外侧部分 22，其中心部分 21的厚度低于外侧部分 22，当活动挡板 2组合在一起时， 会形成一个中间有凹槽的圆盘； 该活动挡板 2的外缘端面具有一凹槽， 该凹槽与密封圈 4的尺寸匹配， 可以使密封圈 4套在上面， 防止滑脱和移位； 活动挡板 2的中心部分 21 与外侧部分 22的交界线上， 即所有活动挡板组合在一起时所形成的凹槽的边缘上设有 三角形缺口 （如图 17所示）， 该三角形缺口能够与多边形部件 11 的顶角相匹配， 当处 于打开状态时， 多边形部件 11的顶角能够落入活动挡板 2内缘上的缺口中 （如图 18所 示）；
而多边形部件 11也相对于实施例 1中的多边形部件 11要薄一些， 其尺寸正好与活 动挡板 2组合在一起形成的圆盘中间的凹槽相匹配，该套管的其他结构与实施例 4相同。
实施例 6
本实施例提供了一种用于生物样本体外纯化处理的离心管，其包括一普通离心管本 体和实施例 1、 2、 3、 4或 5提供的套管， 该套管位于离心管本体之中并与其活动套配。
活动挡板 2具有闭合和打开两种状态， 分别如图 3和图 4所示， 其中： 当处于闭合 状态时， 六块活动挡板 2相互合并在一起与多边形部件 11组成一个圆形， 密封圈 4此 时也处于收缩状态， 套在管体 1第二开口 9的外侧并与活动挡板 2的外缘接触， 管体 1 底部的第一开口 7和活动挡板 2上的第三开口 10不存在重合区域， 管体 1处于一个相 对封闭的状态； 通过旋转同轴锁定开关 5， 可以带动连接轴 3以及多边形部件 11转动， 多边形部件 11转动时其侧边会推动活动挡板 2使其沿半径方向向外运动， 由闭合状态 转换到打开状态， 在该状态下， 活动挡板 2将密封圈 4向外推， 使密封圈 4扩张并接触 到离心管本体的内壁， 将套管与离心管本体之间的缝隙密封， 管体 1底部的第一开口 7 和活动挡板 2上的第三开口 10相互重合， 管体 1处于一个开放的状态， 可以允许位于 套管端面上下两侧的液体相互接触。
实施例 7
本实施例提供了一种用于生物样本体外纯化处理的方法，其是采用实施例 6所提供 的离心管对血液样本中的单个核细胞进行富集的方法， 其流程如图 19所示。 该方法包 括以下步骤：
将套管置于离心管本体内部， 打开套管的活动挡板 2;
将密度介质装入离心管本体中， 装入密度介质的液面达到与套管底端面接触的高 度， 关闭套管的活动挡板 2;
将血液样本直接倾倒入套管中， 无需沿套管内壁小心注入；
打开套管的活动挡板 2， 使血液样本与密度介质接触， 在 20°C、 400g离心力的条件 下进行离心处理， 离心 30分钟之后， 在套管的活动挡板 2上方形成白膜层， 即单个核 细胞层；
关闭套管的活动挡板 2， 将套管从离心管本体中取出， 将富含血小板和细胞碎片的 血浆层用巴斯德吸管或移液管吸取移出， 并将白膜层转移， 完成血液样本中的单个核细 胞的富集。

Claims

权利要求书
1、 一种用于生物样本体外纯化处理的套管， 其包括管体、 若干块活动挡板、 连接 轴、 密封圈、 同轴锁定开关， 其中：
所述管体为筒状， 其上端具有外向的凸缘， 其底部端面上设有若干个第一开口， 其 侧壁的底端设有若干个第二开口以及若干个外向的凸块， 其中， 所述凸块位于相邻两个 所述第二开口的连接部的上方；
所述密封圈套于所述第二开口处的外侧；
所述活动挡板设于所述管体的底部， 所述活动挡板上设有若干个第三开口， 所述活 动挡板的外缘与所述第二开口一一对应并与所述密封圈接触；
所述连接轴位于所述管体的中心位置， 其顶端与所述同轴锁定开关连接， 底端设有 一多边形部件， 该多边形部件的每一条边与一个所述活动挡板的内缘一一对应并接触； 所述同轴锁定开关位于所述管体的凸缘的上表面上并且能够沿该上表面转动； 当所述活动挡板处于打开状态时， 所述第一开口和第三开口之间至少部分重合； 当 所述活动挡板处于闭合状态时， 所述第一开口和第三开口之间不存在重合的部分， 所述 第二开口的形状和尺寸与所述活动挡板的外缘相匹配。
2、 根据权利要求 1所述的套管， 其中， 所述密封圈的粗细与所述第二开口的高度 相同。
3、 根据权利要求 2所述的套管， 其中， 所述密封圈为有弹性的密封圈。
4、 根据权利要求 3所述的套管， 其中， 所述密封圈为橡胶圈。
5、 根据权利要求 1所述的套管， 其中， 所述活动挡板为环扇形， 并且， 所述活动 挡板的厚度与所述多边形部件的厚度相同，所有活动挡板与所述多边形部件组成一个完 整的圆盘形； 或者所述活动挡板为扇形， 所述挡板的中心部分的厚度低于外侧部分的厚 度， 并且， 所述活动挡板的中心部分的厚度与所述多边形部件的厚度之和等于或大于所 述活动挡板外侧部分的厚度， 所有活动挡板与所述多边形部件组成一个完整的圆盘形。
6、 根据权利要求 1所述的套管， 其中， 该套管包括 3-12个活动挡板， 所述第二开 口的数量、 所述凸块的数量、 所述多边形部件的边数与所述活动挡板的数量相同。
7、 根据权利要求 2所述的套管， 其中， 所述活动挡板的外缘侧面具有一与密封圈 相配合的凹槽。
8、 根据权利要求 1所述的套管， 其中， 所述套管的凸缘上设有至少两对锁定槽， 所述同轴锁定开关包括一横杆， 该横杆中间与连接轴顶端连接并能够带动连接轴转动， 其两端分别位于一对锁定槽之中并能够在不同的锁定槽之间转换。
9、 根据权利要求 1所述的套管， 其中， 所述第二开口为矩形。
10、 根据权利要求 1所述的套管， 其中， 所述管体的底部端面上设有第一通孔， 所 述活动挡板为扇形， 其圆心处具有一弧形缺口， 所述挡板的中心部分的厚度低于外侧部 分的厚度，所述活动挡板的中心部分的厚度与所述多边形部件的厚度之和等于或大于所 述活动挡板外侧部分的厚度， 所有活动挡板与所述多边形部件组成一个完整的圆盘形， 所有所述活动挡板的弧形缺口组合在一起构成一个与所述第一通孔对应的第二通孔； 所述多边形部件的底部中心位置具有一柱状部件， 该柱状部件穿过所述第二通孔和 所述第一通孔， 该柱状部件的末端为一卡止端， 该卡止端将所述连接轴和多边形部件纵 向固定。
11、 根据权利要求 10所述的套管， 其中， 所述卡止端的水平尺寸大于所述第一通 孔的直径。
12、 根据权利要求 1所述的套管， 其中， 所述活动挡板的内缘设有与所述多边形部 件的角相匹配的缺口。
13、 根据权利要求 12所述的套管， 其中， 所述活动挡板的内缘的缺口为三角形或 半圆形。
14、 一种用于生物样本体外纯化处理的离心容器， 其包括权利要求 1-13 任一项所 述的用于生物样本体外纯化处理的套管和离心容器本体， 其中， 所述套管位于离心容器 本体之中并且二者活动套配。
15、 根据权利要求 14所述的离心容器， 其中， 所述离心容器为离心管或离心瓶。
16、 根据权利要求 14所述的离心容器， 其中， 当所述活动挡板处于打开状态时， 所述密封圈扩张并能够将所述离心容器本体与所述套管之间的缝隙密封。
17、 一种用于生物样本体外纯化处理的方法， 其是釆用权利要求 14-16任一项所述 的离心容器对生物样本中的生物颗粒进行富集， 其包括以下步骤：
将套管装配在离心容器中， 并打开套管的活动挡板；
将密度介质装入离心容器本体中， 并关闭套管的活动挡板；
将生物样本注入套管中；
打开套管的活动挡板， 使生物样本与密度介质接触， 进行离心处理， 离心处理之后 在套管的活动挡板上方形成生物颗粒富集层；
关闭套管的活动挡板， 将套管从离心容器本体中取出， 将生物颗粒富集层转移， 完 成生物样本中生物颗粒的富集。
18、 根据权利要求 17所述的方法， 其中， 装入离心容器本体中的密度介质的上表 面与所述套管的底端面接触或略高于底端面。
PCT/CN2013/088668 2012-12-05 2013-12-05 一种用于生物样本体外纯化处理的套管 WO2014086302A1 (zh)

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