CN106062170B - For collecting instrument, the system and method for target material - Google Patents

For collecting instrument, the system and method for target material Download PDF

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Publication number
CN106062170B
CN106062170B CN201580011850.4A CN201580011850A CN106062170B CN 106062170 B CN106062170 B CN 106062170B CN 201580011850 A CN201580011850 A CN 201580011850A CN 106062170 B CN106062170 B CN 106062170B
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primary containers
target material
collector
processing container
subfraction
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CN106062170A (en
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L·尤伦
D·E·坎普顿
J·J·诺德伯格
S·卡雷
J·E·伦特
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Rarecyte Inc
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Rarecyte Inc
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    • 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
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/56—Labware specially adapted for transferring fluids
    • B01L3/563—Joints or fittings ; Separable fluid transfer means to transfer fluids between at least two containers, e.g. connectors
    • B01L3/5635—Joints or fittings ; Separable fluid transfer means to transfer fluids between at least two containers, e.g. connectors connecting two containers face to face, e.g. comprising a filter
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C12M47/04—Cell isolation or sorting
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/02—Adapting objects or devices to another
    • B01L2200/026—Fluid interfacing between devices or objects, e.g. connectors, inlet details
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0689—Sealing
    • 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
    • B01L2300/0672—Integrated piercing tool
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0409—Moving fluids with specific forces or mechanical means specific forces centrifugal forces
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L9/00—Supporting devices; Holding devices
    • B01L9/06—Test-tube stands; Test-tube holders
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G01N1/4077—Concentrating samples by other techniques involving separation of suspended solids
    • G01N2001/4083—Concentrating samples by other techniques involving separation of suspended solids sedimentation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
    • G01N2035/1027—General features of the devices
    • G01N2035/1048—General features of the devices using the transfer device for another function
    • G01N2035/1053—General features of the devices using the transfer device for another function for separating part of the liquid, e.g. filters, extraction phase

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
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  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Microbiology (AREA)
  • Sustainable Development (AREA)
  • Analytical Chemistry (AREA)
  • Cell Biology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Hematology (AREA)
  • Sampling And Sample Adjustment (AREA)
  • Centrifugal Separators (AREA)

Abstract

This disclosure relates to the instruments for recycling target material from suspension, system and method.System includes multiple processing containers and collector.Collector, which injects the part of the target material from suspension with funnel, to be passed through intubation and enters in processing container.Sequential density classification is by gradually or sample is divided into fraction or sample fraction is divided into subfraction by sequential process, so that each step or sequence cause from previously and the collection of subsequent step or the different fractions or subfraction of sequence or separated.In other words, sequential density classification provides each subgroup of group or each sub- subgroup of group's subgroup by series of steps.

Description

For collecting instrument, the system and method for target material
With the cross reference of related application
This application claims the interests for the U.S. Provisional Patent Application No. 61/935,457 submitted for 4th for 2 months in 2014, and And the application is also the part continuation application for the U.S. Patent Application No. 14/495,449 submitted for 24th in September in 2014, it is described U.S. Patent Application No. 14/495,449 is the U.S. Patent Application No. 14/090,337 submitted on November 26th, 2013 Part continuation application, the U.S. Patent Application No. 14/090,337 are required in the United States Patent (USP) submitted on November 30th, 2012 Provisional Application No. 61/732,029；In the U.S. Provisional Patent Application No. 61/745,094 that on December 21st, 2012 submits；In The U.S. Provisional Patent Application No. 61/791,883 that on March 15th, 2013 submits；In on May 1st, 2013 U.S. submitted it is special Sharp Provisional Application No. 61/818,301；With the U.S. Provisional Patent Application No. 61/869,866 submitted for 26th in August in 2013 Interests；And the application is also that Shen is continued in the part for the U.S. Patent Application No. 14/266,939 submitted on May 1st, 2014 Please, the U.S. Patent Application No. 14/266,939 is required in the U.S. Provisional Patent Application No. submitted on May 1st, 2013 61/818,301, in the U.S. Provisional Patent Application No. 61/869 that August in 2013 is submitted on the 26th, 866, and in 2 months 2014 4 The interests for the U.S. Provisional Patent Application No. 61/935,457 that day submits.
Technical field
Present disclosure relates generally to the fluid based on density and separates, and target material is in particular to recycled from suspension.
Background
Suspension generally includes to be difficult to detect, extracts and separate the target material for analysis.For example, whole blood is material Suspension in a fluid.Material include billions of red blood cells in the protein fluid of referred to as blood plasma and leucocyte and Blood platelet.Presence with regard to aberrant biological or cell carries out routine examination to whole blood, the aberrant biological or cell such as ovum, Fetal cell, endothelial cell, helminth, bacterium and inflammatory cell and virus include HIV, cytomegalovirus, hepatitis C Virus and Epstein-Barr virus.Currently, practitioner, researcher and with blood sample work those of people attempt separate, separation and extract peripheral blood Certain components of sample are for checking.For analyzing the usual technology of blood sample the following steps are included: by blood film smear in glass slide On, and dyeing film in this way, certain components are checked by bright-field microscopy art.
On the other hand, it is difficult to (if impossible) in suspension with the target material that extremely low concentration occurs It is detected and is analyzed using many prior arts.Consider such as circulating tumor cell (" CTC "), be from tumour be detached from, The cancer cell of the seed of the subsequent growth for the other tumour that recycles, and can be considered as in different tissues in blood flow (turns It moves).The ability of accurate detection and analysis CTC are that oncologist and cancer research person are of special interest.However, CTC is with pole Low number appears in peripheral blood sample.For example, containing as little as 5 CTC peripheral blood sample 7.5ml sample be considered as The diagnosing and treating of cancer patient is clinically relevant.In other words, 5 CTC detected in 7.5ml blood sample are equivalent to detect 1CTC in the background of about 10,000,000,000 red blood cells and leucocyte, this is extremely time-consuming, expensive using blood film analysis and is difficult to reality Existing.
Therefore, practitioner, researcher and with suspension work those of people continue to look for the presence with regard to rare target material Or be not present and the system and method for accurately analyzing suspension.
Attached drawing description
Figure 1A -1B shows example collector.
Fig. 2A -2B shows example collector.
Fig. 2 C-2D shows example collector.
Fig. 3 A-3B shows example collector-processing containment system.
Fig. 4 A-4B shows example collector-shelter (canopy) system.
Fig. 5 A-5B shows example seal ring.
Fig. 5 C-5D shows example seal ring.
Fig. 5 E-5F shows example seal ring.
Fig. 5 G shows example seal ring.
Fig. 6 shows the flow chart of the exemplary method for recycling target material.
Fig. 7 A-7B shows example float and Primary containers system.
Fig. 8 shows the separated example float and Primary containers system undergone based on density.
Fig. 9 shows to form the example seal ring of sealing and example float and Primary containers system.
Figure 10 A-10G shows the example system of recycling target material.
It is described in detail
This disclosure relates to the instruments for recycling target material from suspension, system and method.System includes multiple Process container and collector.Collector injects the part of the target material from suspension funnel in processing container.It is sequential close Degree classification is by gradually or sample is divided into fraction or sample fraction is divided into subfraction by sequential process, so that each step Or sequence leads to the collection or separated from previous and sequential step or sequence different fractions or subfraction.In other words, sequence It passes through density classification and provides each subgroup of group or each sub- subgroup (sub-sub- of group's subgroup by series of steps population)。
Collector
Figure 1A shows the isometric view of collector 100.Figure 1B shows the receipts that the line I-I shown in Figure 1A is obtained The viewgraph of cross-section of storage 100.Chain-dotted line 102 represents center or the highest symmetry axis of collector 100.Collector 100 can have It is of such dimension that and shape, to fit in containing or can accommodate in the Primary containers of suspension, the suspension is under a cloud Including target material.Target material from suspension funnel by 106 injection of intubation and is entered processing container by collector 100 In (not shown), to be located in chamber 108.Collector 100 includes main body 104 comprising first end 110 and the second end 112.Sealing can be formed between the second end 112 and the inner wall of Primary containers, with before centrifugation, centrifugation during and be centrifuged The sealing engagement of fluid-tight is maintained afterwards, and any part of suspension is inhibited to be located at the inner wall and collector of Primary containers Between 100 main body 104, or flow between the inner wall of Primary containers and the main body 104 of collector 100.Sealing can pass through Following to be formed: interference engagement, adhesive, epoxy resin, (such as is passed through by bonding lubricating grease (such as vacuum grease) Hot adhesion), by welding (such as passing through ultrasonic bonding), by clamping (such as with ring or fixture), fitting in second end Insertion piece (such as O-ring or lantern ring) between portion 112 and the inner wall of Primary containers etc..Main body 104 can be any appropriate Shape, including but not limited to cylinder, triangle, square, rectangle etc..Collector 100 further includes internal funnel 114, For concave-shaped openings.Funnel 114 can be tapered from the second end 112 towards intubation 106.Funnel 114 will be from lower than the The target material of two ends 112 is directed in intubation 106, and the intubation 106 connect and is in fluid communication with the vertex of funnel 114.Leakage The vertex of bucket 114 has the oral area smaller diameter than funnel 114.Funnel 114 passes through the wall that is tapered and is formed, it is described by The thin wall of gradual change can be straight, curve, bowed etc..Funnel 114 can be any suitable shape, including but not It is limited to tubular, spherical, cheese, cone, rectangle, pyramid etc..In addition, the most external diameter or edge of funnel 114 It can be continuously connected to the inner wall of Primary containers or constant contact (flushing), so that in the second end 112 of collector 100 Idle space is not present between the inner wall of Primary containers.
Intubation 106 such as pipe or needle include but is not limited to non-coring needle, extend from the vertex of funnel 114 and enter chamber 108 It is interior.In the example in fig 1, chamber 108 is the concave-shaped openings extended into main body 104 from first end 110, and can be connect By and support processing container (not shown).Chamber 108 can be any depth appropriate, (not shown with receiving and supporting to process container Out).Intubation 106 can extend any appropriately distance and enter in chamber 108, to penetrate the base portion of processing container (not shown), Or in insertion processing container.Intubation 106 may include flat tip, chamfered tip, sharp tip or cone point.In addition, chamber 108 can be any proper shape, including but not limited to tubular, spherical, cheese, cone, rectangle, pyramid etc. Deng.Chamber 108 can be it is threaded, with engage processing container threaded section (not shown).
Collector 100 can also include retainer (not shown), to prevent collector 100 from sliding relative to Primary containers, To make collector 100 be maintained at the predetermined height in Primary containers.Retainer (not shown) can be from first end 110 shoulder, fixture, the annular projections of circumference for extending beyond cylinder-shaped body 104, retainer etc. radially extended.
Fig. 2A shows the isometric view of collector 200.Fig. 2 B shows what the line II-II shown in Fig. 2A was obtained The viewgraph of cross-section of collector 200.Chain-dotted line 202 represents center or the highest symmetry axis of collector 200.Collector 200 is similar In collector 100, in addition to collector 200 includes the main body 204 more elongated than the main body of collector 100, hold to accommodate processing Except the greater portion of device (not shown).Main body 204 includes first end 206 and the second end 208.Sealing can be second Formed between end 208 and the inner wall of Primary containers, with before centrifugation, centrifugation during and centrifugation after maintain fluid-tight it is close Sealing-in is closed, and any part of suspension is inhibited to flow between the inner wall of Primary containers and the main body 204 of collector 200. Sealing can be formed by following: interference engagement, lubricating grease (such as vacuum grease), adhesive, epoxy resin, bonding (such as hot adhesion), by welding (such as passing through ultrasonic bonding), clamp (such as with ring or fixture), fit in second end Insertion piece (such as O-ring or lantern ring) between portion 208 and the inner wall of Primary containers etc..
First end 206 includes having a size of at least part of chamber 212 for receiving and accommodating processing container (not shown). Chamber 212 can have the taper or stepped bottom end 220 that processing container (not shown) can be positioned on.First end Portion 206 can also include at least one notch (cut-out) 210, be used for allowing to process the appropriate grasping of container (not shown) It is inserted and removed from.Collector 200 injects the target material from suspension at the second end 208 with funnel by intubation 214 Inside funnel 222 in, and enter be located at chamber 212 in processing container (not shown) in.Intubation 214 can be placed in frame 224 On, so that the internal holes of intubation 214 are flushed with the inner wall of funnel 222, as shown in Figure 2 B.
Collector 200 may include shoulder 216, and the shoulder 216 circumference around main body 204 extends.Shoulder 216 can To be longer than the internal diameter of Primary containers, to be placed in the open end of Primary containers, and to Primary containers and shoulder After 216 external application locking ring (not shown), to inhibit collector 200 relative to the movement of Primary containers.Locking ring is (not Show) pressure is applied to Primary containers along shoulder 216.Locking ring can be two rings, the whole circumference week in Primary containers Enclose a loop of package or the loop that wraps up around whole circumference that is less than in Primary containers, such as half (1/2), 5/8ths (5/8), 2/3rds (2/3), 3/4ths (3/4), 7/8ths (7/8) etc..Alternatively, shoulder 216 can To fit in Primary containers.Alternatively, shoulder 216 can be fixture, so that shoulder 216 may include that Primary containers can be with The clamping lock being inserted into it, to inhibit collector 200 relative to the movement of Primary containers.Alternatively, shoulder 216 can with it is main The inside of container forms interference engagement, and sealing ring can be placed in around it.
As shown in Figure 2 A, collector 200 may include at least one window 218, with by the inner wall of main body 204 into Enter (access) chamber 212.At least one window 218 allows operator to confirm that processing container (not shown) is appropriate in chamber 212 It places.At least one window 218 also allows to remove fluid from intubation 214, to flow out collector 200 and enter in collector It is formed between 200 and Primary containers (not shown), and in the sealing between the second end 208 and the inner wall of Primary containers In the space of side.
Fig. 2 C shows the isometric view of collector 230.Fig. 2 D shows that the line III-III shown in Fig. 2 C is obtained Collector 230 viewgraph of cross-section.Collector 230 is similar to collector 200, in addition to collector 230 includes main body 238, institute Stating main body 238 includes the prolongation 234 for extending outwardly away from first end 232 and at least opening in temporarily sealing prolongation 234 The lid 236 of mouth 240.Opening 240 can be in fluid communication at first end 232 with chamber 212.Lid 236 can be it is removable, can Penetrate with reclosable (such as hinged cover), or can penetrate and not reclosable (such as foil lid).Prolongation 234 can To have the size for receiving lid 236 when penetrating, so that a part of lid 236 can not extend at first end 232 In chamber 212.It should be noted that collector 230 does not include at least one notch 210.
Main body can be made of various different materials, and the various different materials include but is not limited to ceramics；Metal；Have Machine or inorganic material；With plastic material such as polyformaldehydePolystyrene, acronitrile-butadiene-styrene (" ABS ") copolymer, aromatic polycarbonate, aromatic polyester, carboxymethyl cellulose, ethyl cellulose, ethylene vinyl acetate are total Polymers, nylon, polyacetals, poly- acetic acid esters, polyacrylonitrile and other nitrile resins, acrylonitrile-vinyl chloride copolymer, polyamides Amine, aromatic polyamides (" aramid "), polyamide-imides, polyarylate, polyarylene oxide, poly arylidene thio-ester, poly- virtue Base sulfone, polybenzimidazoles, polybutylene terephthalate (PBT), polycarbonate, polyester, polyesterimide, polyether sulfone, polyethers acyl Imines, polyether-ketone, polyether-ether-ketone, polyethylene terephthalate, polyimides, polymethacrylates, polyolefin are (such as poly- Ethylene, polypropylene), polyallomer (polyallomers), polyoxadiazoles, Parylene, polyphenylene oxide (PPO), MODIFIED PP O, Polystyrene, polysulfones, fluoropolymer for example polytetrafluoroethylene (PTFE), polyurethanes, polyvinylacetate, polyvinyl alcohol, Polyvinyl halide such as polyvinyl chloride, polyvinylpyrrolidone, gathers inclined dichloro at polyvinyl chloride-acetate copolymer Ethylene, special copolymer, polystyrene, polycarbonate, polypropylene, acrylonitrilebutadienestyrene copolymer, butyl rubber, Ethylene propylene diene monomer；And combinations thereof.
Intubation can be made of various different materials, and the various different materials include but is not limited to ceramics；Metal；Have Machine or inorganic material；With plastic material such as polypropylene, acrylic acid, polycarbonate etc.；And combinations thereof.Intubation can have Along the tip of the intubation longitudinal axis.
Collector-processing containment system
Fig. 3 A shows example collector 200 and processes the decomposition view of container 302.Fig. 3 B shows the institute along Fig. 3 A What the line IV-IV shown was obtained, the transversal of the processing container 302 in chamber 212 is inserted at the first end 206 of collection vessel 200 Face view.Collector 200 and processing container 302 form collector-processing containment system 300.Processing container 302 can be Eppendorf pipe, syringe or test tube, and there is closure end 304 and open end 306.Open end 306, which has, to be connect By the size of cap 308.Cap 308 can be made of resealable rubber or other suitable resealable materials, it is described can be close again Closure material can be pierced through repeatedly with needle or other sharp instruments, to approach the content stored in processing 302 inside of container, and And it is sealed again when needle or utensil remove.Alternatively, processing container 302 can also have two openings of the size for receiving cap End.Processing container 302 can have the taper geometry widened or narrowed towards open end 306；Process container 302 It can have substantial cylindrical geometry；Alternatively, processing container 302 can have it is substantial cylindrical in the first section Geometry and conical geometry in the second section, wherein the first section and the second section be connected to each other and even It is continuous.Although at least one section for processing container 302 has circular cross section, in other embodiments, at least one area Section can have ellipse, square, triangle, rectangle, octagon or any other suitable cross-sectional shape.Processing is held Device 302 can be made of transparent, translucent, opaque or semipermeable materials such as plastics or another suitable material.Processing Container includes central axis 314, and when being inserted into chamber 212, the central axis 202 of the central axis 314 and collector 200 is coaxial.Add Chemical container 302 can also include the plug 310 at closure end 304, to allow the introducing of target material or make target material and displacement Fluid 312 exchanges.Closure end 304 can be it is threaded, to provide the screw thread with the threaded chamber 212 of collector 200 Connection.Processing container 302 can be made of glass, plastics or other suitable materials.
Plug 310 can be made of resealable rubber or other suitable resealable materials, the resealable material It can be pierced through repeatedly with needle or other sharp instruments, to approach the content inside processing container 302 or content is allowed to introduce It processes in container 302, and is sealed again when needle or utensil remove.Plug 310 can be inserted in processing container 302, so that sealing Such as it is maintained between plug 310 and processing container 302 by interference engagement.Alternatively, heating liquid rubber can be used in plug 310 Glue is formed in the closure end 304 of processing container 302, and the heating liquid rubber can shape and work as in heating or heat Rubber hardens when cooling.Adhesive can be used for fill in 310 inner walls for being attached to processing container, can be based on polymer Adhesive, epoxy resin, contact adhesive or any other suitable material for bonding or generating hot adhesion.Alternatively Ground, plug 310 can be mapped in processing container 302.Alternatively, plug 310 can be with processing 302 hot adhesion of container.
In the example of Fig. 3 B, intubation 214 has cone point, penetrates plug 310 and extends into processing container 302 Inner chamber, wherein intubation 214 handle do not extend into processing container 302 inner chamber.As described in more detail below, The inner chamber for processing container 302 accommodates target material.Intubation 214 can be covered by resealable sleeve (not shown), to prevent target Material outflow, except non-processing container 302 in chamber 212 to allow be intubated 214 just penetrate processing container 302 depth.It can Seal sleeve (not shown) covering intubation 214 again, is spring, can be penetrated by intubation 214, and by that can be subjected to It firmly repeats to pierce through that the elastomeric material of sealing is still maintained to be made simultaneously.
As shown in Fig. 3 A-3B, before being inserted into collector 200, processing container 302 can be mounted with displacement fluid 312.Displacement fluid 312 replaces target material, so that when collector 200 and the processing insertion of container 302 include target material Primary containers In (not shown), and when collector, processing container and Primary containers experience centrifugation, the outflow processing container of displacement fluid 312 302 and enter Primary containers in, and by displacement for example pass through buoyancy displacement (that is, being lifted up material), push target Material passes through intubation 214 and enters in processing container 302.
Displacement fluid 312 has the density bigger than the density of the target material of suspension, and (density can be greater than suspension grade The density of the subset or all suspension fractions divided), and be inert for suspension material.Displacement fluid 312 is suspending It can be in fluid miscible or not miscible.The example of suitable displacement fluid includes but is not limited to be coated with poly- second Colloidal silica particle solution (such as Percoll), the polysaccharide solution (such as Ficoll), Iodixanol of alkene pyrrolidone (such as OptiPrep), organic solvent, liquid wax, oil, gas and combinations thereof；Olive oil, mineral oil, silicone oil, immersion oil, Mineral oil, paraffin oil, silicone oil, fluorosilicone, perfluorodecalin, perfluor perhydro phenanthrene, perfluoroctylbromide and combinations thereof；Have Solvent such as 1,4- dioxanes, acetonitrile, ethyl acetate, the tert-butyl alcohol, cyclohexanone, methylene chloride, tert-pentyl alcohol, methyl tertbutyl Ether, butyl acetate, hexanol, nitrobenzene, toluene, octanol, octane, propene carbonate, sulfolane and ionic liquid；Based on polymerization The solution of object；Surfactant；It is perfluor ketone such as perfluorocyclopentanone and perfluorocyclohexanone, fluorinated ketone, hydrofluoroether, hydrofluorocarbon, complete Fluorocarbons, perfluoropolyether, silicon and the liquid such as phenylmethylsiloxane based on silicon；And combinations thereof.
Processing container 302 can also include processing solution (not shown), real with when target material enters processing container 302 Now to the conversion of target material.Processing solution (not shown) can be preservative, cell adherence solution, dyestuff etc..It is flowed with displacement Body 312 is different, and most of (and if not all) processing solution (not shown) are retained in after centrifugation in processing container 302, To in one way or another way (i.e. anti-corrosion, increase adhesion characteristics etc.) realizes conversion to target material.It processes molten Liquid (not shown) can be used as the liquid for including in liquid or shell and introduce.Shell can be dissolved in aqueous solution rather than replace In fluid 312 (such as gel cap)；Or shell can be it is frangible so that when processing container 302 vibrated in turbine mixer When, outer casing rupture.Further, it is possible to use being more than a kind of processing solution.
Processing container 302 may include flexible cap, can be pushed will be assigned to matrix example by its predetermined volume On glass slide or orifice plate.Cap 308 can be flexible or cap 308 can be removed, and flexible cap is inserted into open end In portion 306.Alternatively, processing container 302 can be attached to distributor (i.e. after gathering target material), or may include distribution The target material of predetermined volume can be assigned to another matrix such as microscope from processing container 302 by device, the distributor On glass slide.Distributor can repeat to pierce through the material in resealable cap 308 or compression process container 302, to extract and The target of distribution predetermined volume is expected in matrix.Alternatively, cap 308 can be removed, and distributor (not shown) can be straight It patches in processing container 302, to distribute buffy coat-processing solution mixture.
Collector-shelter system
Fig. 4 A shows the decomposition view of example collector 200 and shelter 402.Fig. 4 B is shown shown in Fig. 4 A What line V-V was obtained, it is inserted into the viewgraph of cross-section of the processing container 402 in the chamber 212 of collector 200.Collector 200 and shelter 402 form collector-shelter system 400.Shelter 402 is similar to processing container 302, in addition to shelter has the second open end Except 404.When collector-shelter system 400 is inserted into Primary containers, some fluids in Primary containers such as one Divide suspension, a part of suspension fraction, a part of cleaning fluid etc., 214 discharge of intubation can be passed through.Shelter 402 inhibits The opening of the first end 206 of collector 200 can be escaped from by a part of fluid in the Primary containers of 214 discharge of intubation. Pass through the discharge fluid that shelter 402 blocks and flowed out the second open end 404, and flows out window 218.The display of dotted line 406 is worked as Fluid flowing when fluid is by 214 discharge of intubation, and retained by shelter 402.
Alternatively, when using collector 230, the lid 236 of collector 230 inhibits can be by the master of 214 discharge of intubation A part of fluid in container is wanted to escape from the opening of the first end 206 of collector 200, it's similar to those of shelter 402 Kind.
Sealing ring
Fig. 5 A shows the isometric view of sealing ring 500.Fig. 5 B show sealing ring 500 from the lower view in top.Click and sweep Line 502 represents center or the highest symmetry axis of sealing ring 500.Sealing ring 500 includes inner wall 504, outer wall 506 and chamber 508.In In Fig. 5 B, RIWIt represents from the center of sealing ring 500 to the radial distance of inner wall 504, and ROWIt represents from sealing ring 500 Radial distance of the center to outer wall 506.Sealing ring 500 is configured to fit in around Primary containers such as pipe.Chamber 508, which has, to be connect By the size and shape of Primary containers.Sealing ring 500 can screw so that by around outer wall 506 circumference apply guiding it is close The substantially uniform radial force of the central axis 502 of seal ring 500, such as the radial force generated by fixture, the size of chamber 508 with And the radius of inner and outer wall 504 and 506 is reduced.When sealing ring 500 screws around Primary containers, it is applied to sealing ring 500 uniform force is applied to Primary containers, so that Primary containers be promoted to shrink.It is close when radial force is removed from sealing ring 500 Seal ring 500 keeps screwing and in tension around Primary containers.
Sealing ring can be any shape, including but not limited to round, triangle or polyhedron.Fig. 5 C shows sealing The isometric view of ring 510.Fig. 5 D show sealing ring 510 from the lower view in top.Sealing ring 510 is similar to sealing ring 500, Other than sealing ring 510 is polyhedron.Chain-dotted line 512 represents center or the highest symmetry axis of sealing ring 510.Sealing ring 510 Including inner wall 514, outer wall 516 and chamber 518.Sealing ring can be by metal such as brass, polymer or combinations thereof.
Alternatively, as shown in fig. 5e, sealing ring 520 can be made of piezoelectric material.Fig. 5 F shows sealing ring 520 From the lower view in top.Chain-dotted line 522 represents center or the highest symmetry axis of sealing ring 520.Sealing ring 520 can be via One conducting wire 524 and the second conducting wire 526 are attached to potential source 528, such as battery.The generation of potential source 528 promotes sealing ring 520 to twist The tightly mechanical stress of (i.e. 520 radius of sealing ring is reduced).Sealing ring 520 includes inner wall 530, outer wall 532 and chamber 534.Scheming In 5F, RIWIt represents from the center of sealing ring 520 to the radial distance of inner wall 530, and ROWIt represents from the center of sealing ring 520 To the radial distance of outer wall 532.Alternatively, sealing ring 520 may be at natural tight condition.When an electrical potential is applied, it seals Ring 520 is expanded.Alternatively, a part of of sealing ring can be made of piezoelectric material, so that piezoelectric serves as driver, with Promote the other parts of sealing ring to tighten and substantially uniform circumferential pressure is applied to Primary containers, to make Primary containers It shrinks to form sealing.
Fig. 5 G shows the isometric view of sealing ring 540.Sealing ring includes adjustment mechanism 548, to adjust internal diameter RID.It can Puckered ring includes first end 542 and the second end 546, and the first end 542 and the second end 546 pass through band part 544 Connection.First end 542 and the second end 546 include the complementary portion of adjustment mechanism 548.Adjustment mechanism 548 includes but unlimited In ratchet, tongue-and-groove, pawl etc..
Sealing ring can also include temperature-sensitive element, such as heater wire.Temperature-sensitive element can soften Primary containers for receiving Contracting.Alternatively, temperature-sensitive element can melt Primary containers, to provide the sealing more adhered to.Alternatively, temperature-sensitive element can promote Compress sealing ring, to form sealing between Primary containers and float.
Sequential density step-procedure
Sequential density classification be by gradually or sample is divided into fraction to sequential process or sample fraction is divided into subfraction, So that each step or sequence cause from previously and sequential step or sequence different fractions or subfraction collection or divide It opens.In other words, sequential density classification provides each subgroup of group or each Asia Asia of group's subgroup by series of steps Group.For example, buffy coat is the fraction of whole blood sample.Buffy coat fraction can further decompose into subfraction, packet Include but be not limited to granulophilocyte, granulocyte, lymphocyte/monocyte and blood platelet.These subfractions can pass through execution The sequential each acquisition of density classification.
For convenience, this method is described with reference to the example flyout liquid of anticoagulated whole blood.But method described below does not have It is directed in its application range and is so limited.In practice, this method can be used for any kind of suspension.Example Such as, sample suspension can be urine, blood, marrow, cyst fluid, ascites, excrement, sperm, celiolymph, nipple aspirate fluid, saliva Liquid, amniotic fluid, vaginal fluid, mucosal secretion, aqueous humor, vitreous humor, vomitus and any other physiological fluid or half Solid.It should also be understood that target material can be the fraction of sample suspension, such as buffy coat, cell such as ovum, Erythroblast, foetal DNA, fetal rna etc. or circulating tumor cell (" CTC "), circulating endothelial cells, immunocyte (i.e. initial or memory B cell or initial or memory T cell), liposome, protein, nucleic acid, biomolecule, has vesica Close membrane naturally occurring or the microscopic units manually prepared, helminth (such as conveyor screw, such as cause the Bu Shi of Lyme disease Borellia (Borrelia burgdorferi)；Malaria (malayria) inducer), microorganism, virus or inflammatory cell. Alternatively, sample can be biosolids and for example organize, before or after Primary containers are added, biosolids example Such as decomposed by clostridiopetidase A.
Fig. 6 shows the flow chart for being used to recycle the exemplary method of target material using sequential density classification.In box 602 In, obtain suspension such as anticoagulated whole blood.In box 604, whole blood is added in Primary containers such as test tube.Float can also To be added in Primary containers.For convenience, this method is described for float, but methods described below does not have purport It is so being limited in its application, and may not need float and execute.
Fig. 7 A shows the isometric view of example Primary containers and float system 700.System 700 includes Primary containers 702 With the float 704 being suspended in whole blood 706.In the example of Fig. 7 A, there is Primary containers 702 circular cross section, first to open End 710 and the second closure end 708.Open end 710 has the size for receiving cap 712.Primary containers, which can also have, to be connect By two open ends of the size of cap, such as example pipe shown in Fig. 7 B and separable float system 720.System 720 Similar to system 700, other than Primary containers 702 replace with Primary containers 722, the Primary containers 722 include being configured to Receive two open ends 724 and 726 of cap 712 and cap 728 respectively.Primary containers 702 and 722 have substantial cylindrical Geometry, it is also possible to have the taper geometric form widened respectively, narrowed or combinations thereof towards open end 710 and 724 Shape.Although Primary containers 702 and 722 have circular cross section, in other embodiments, Primary containers 702 and 722 can With with ellipse, square, triangle, rectangle, octagon or basic upper extension tube length any other suitably it is transversal Face shape.Primary containers 702 and 722 can be by transparent, translucent, opaque or semipermeable materials such as plastics or another Kind suitable material composition.Primary containers 702 and 722 respectively include central axis 718 and 730.Primary containers 702 can be with It is such as visible in enlarged view 716 including the diaphragm 714 at closure end 708, to allow fluid, suspension or hang The removal of floating fraction either uses syringe, pump, by drainage etc..Primary containers 702 can have inner wall and first directly Diameter.
Diaphragm 714 can be made of resealable rubber or other suitable resealable materials, the resealable material Material can be pierced through repeatedly with needle or other sharp instruments, to enter the content inside Primary containers 702, and work as needle or device It is sealed again when tool removal.Diaphragm 714 can be inserted in Primary containers 702 so that sealing for example by interference engagement maintain every Between film 714 and Primary containers 702.Alternatively, opening and/or bottom of the heating liquid rubber in pipe can be used in diaphragm 714 Portion is formed in inside, and the heating liquid rubber can be shaped in heating or heat and be hardened when rubber is cooling.Adhesive can With the wall for being attached to diaphragm 714 inside opening and pipe, and can be adhesive based on polymer, epoxy resin, Contact adhesive or for by rubber bound to plastics or generate hot adhesion any other suitable material.Alternatively, Diaphragm 714 can be with 702 hot adhesion of Primary containers.
Float 704 includes main body, two tear drop shape end caps and is radially spaced in main body and the branch of axial orientation Support component.Alternatively, float 704 can not include any supporting member.Alternatively, float 704 may include supporting member, Its inner wall for not engaging Primary containers 702.
In alternative embodiment, supporting member number, supporting member interval and support member thickness can be respectively independent Ground changes.Supporting member can also be destroyed or be segmented.Main body is dimensioned such that, to have less than Primary containers 702 The outer diameter of internal diameter, to be limited to the fluid retention channel between the outer surface of main body and the inner wall of Primary containers 702.Support Body surfaces between component can be flat, curved or have another suitable geometry.Supporting member and Main body can be single structure, or can be separate structure.
Embodiment includes the other kinds of geometry shape for float end cap.Top end cap can be tear drop shape, Cheese, cone or any other proper shape.Bottom end cap can be tear drop shape, cheese, cone or it is any its His proper shape.In other embodiments, the main body of float 704 may include various different support constructions for separating Suspension is oriented to around float by sample, support tube wall during centrifugation.Embodiment is not intended to be limited to these Example.Main body may include providing many protrusions of the support for pipe.In alternative embodiment, protrusions number and mode It can change.Main body may include single continuous helical structure or shoulder, and the spiral around main body generates helical duct. In other embodiments, spiral shoulder can be mellow and full or broken or segmentation, to allow fluid in spiral shoulder Adjacent corners between flow.In various embodiments, spiral shoulder interval and rib thickness can be changed independently.Another In one embodiment, main body may include the supporting member that radially from the body and circumference extends around main body.In another reality It applies in scheme, supporting member can be taper.
Float 704 can be made of various different materials, and the various different materials include but is not limited to metal；It is organic Or inorganic material；Iron content plastics；Sintering metal；The metal of machining；Plastic material and combinations thereof.Primary containers 702 can be with With inner wall and first diameter.Float 704 can be captured by being an interference fit in Primary containers 702, so that being centrifuged When, inside pipe wall expands the axial movement to allow float 704.When being centrifuged stopping, inner wall rolls back first diameter, with induction Interference engagement.Alternatively, inner wall can not be expanded, and interference engagement can be between float 704 and Primary containers 702 not Occur, so that float moves freely in pipe before centrifugation, during centrifugation or after centrifugation.The end cap of float can pass through machinery A part based on processing, injection molded, additive technology etc. manufacture, to become a single structure；Alternatively, logical Cross pressing adaptation, adhesive, screw, by its any other method appropriate that at least two panels connects together or combinations thereof, End cap could be attached to main body.
Cap 712 can be made of various different materials, and the various different materials include but is not limited to organic or inorganic material Material；Plastic material；And combinations thereof.
Fig. 6 is returned to, in block 606, Primary containers, float and whole blood experience are for example by being centrifuged point based on density It opens, so that whole blood be allowed to be divided into the fraction based on density along the axial position of pipe based on density.Fig. 8, which is shown, have been undergone for example By the isometric view for being centrifuged the separated Primary containers based on density and float system 700.Assuming that the whole blood packet of such as centrifugation Include three fractions.For convenience, three fractions include blood plasma, buffy coat and red blood cell.However, suspending when another When liquid experience centrifugation, there may be more, less or equal number of fraction, each fraction has different densities.In Fig. 8 Shown, suspension experience is divided into the axially spaced of three fractions along the length of pipe based on density, and wherein red blood cell 803 is located at bottom Portion, blood plasma 801 is located at top, and buffy coat 802 is located between the two.Float 704 can have any appropriate close Degree, to be settled in one of fraction.The density of float 704 can select in this way, so that float 704 is expanded, buffy coat 802 between the main body of float and the inner wall of Primary containers.Buffy coat 802 can be trapped in float 704 and Primary containers In region between 702.
At least one, which divides fluid (delineation fluid) (not shown), may be used to provide target material and in target Being spaced further apart between any non-target material of material above and or below.At least one divides fluid (not shown) can be with With the density for being more than or less than target material.For example, when expectation is spaced further apart buffy coat 802 and red blood cell 803, Dividing fluid can have greater than buffy coat 802 and less than the density of red blood cell 803.At least one divides fluid (not Show) can be can be miscible or not miscible with suspension, it and is inert for suspension material.It is at least one The region for wherein sealing Primary containers 702 can also be provided by dividing fluid (not shown), because in buffy coat 802 and red There is bigger division between cell 803 and separates.At least one division fluid can be used in float whether or not using (not shown).The suitable example for dividing fluid includes but is not limited to the colloidal silica silicon grain for being coated with polyvinylpyrrolidone Sub- solution (such as Percoll), polysaccharide solution (such as Ficoll), Iodixanol (such as OptiPrep), cesium chloride, sucrose, Solution based on sugar, the solution based on polymer, surfactant, organic solvent, liquid wax, oil, gas and combinations thereof；Olive Olive oil, mineral oil, silicone oil, immersion oil, mineral oil, paraffin oil, silicone oil, fluorosilicone, perfluorodecalin, perfluor perhydro are luxuriant and rich with fragrance, complete Fluoro-octane bromide compound and combinations thereof；Organic solvent such as 1,4- dioxanes, acetonitrile, ethyl acetate, the tert-butyl alcohol, cyclohexanone, dichloro Methane, tert-pentyl alcohol, methyl tertiary butyl ether(MTBE), butyl acetate, hexanol, nitrobenzene, toluene, octanol, octane, propene carbonate, ring Fourth sulfone and ionic liquid；Solution based on polymer；Surfactant；Perfluor ketone such as perfluorocyclopentanone and perfluorocyclohexanone, Fluorinated ketone, hydrofluoroether, hydrofluorocarbon, perfluocarbon, perfluoropolyether, silicon and the liquid such as phenylmethylsiloxane based on silicon；And A combination thereof.
Fig. 9 shows the sealing for being formed as that fluid is prevented to move up or down in Primary containers.Sealing also inhibits floating Son is mobile.Sealing ring 500 applies circumference or radial force to Primary containers 702, to promote Primary containers 702 for float 704 It is folded inward.Enlarged view 902 shows the sealing ring 500 tightened around float and Primary containers system 700.It has been placed in The sealing ring 500 of the interface of buffy coat 802 and red blood cell 803 promotes Primary containers 702 to be folded inward, until sealing It is formed between Primary containers 702 and float 704.The outer wall of sealing ring 500 can be flushed with the outer wall of Primary containers 702；It is close The outer wall of seal ring 500 can extend across the outer wall of Primary containers 702；Alternatively, the outer wall of Primary containers 702 can extend more Cross the outer wall of sealing ring 500.The holding of sealing ring 500 is tightened to maintain to seal, this prevents fluid from being moved across in any direction Sealing.Sealing ring 500 may also stay at tension state.Alternatively, sealing ring 500 can be tightened excessively, and then remove The power that sealing ring 500 is applied.Sealing ring 500 can slightly be expanded, although still remaining retracted.
For application sealing ring 500 and to form sealing, fixture can be used for that the center of Primary containers 702 will be directed to The power circumference of axis is applied to sealing ring 500 and float and Primary containers system 700.Float and Primary containers system 700 Experience for example by be centrifuged based on sealing separate after, sealing ring 500 is placed in around float and Primary containers system 700.It is close Seal ring 500 and float and Primary containers system 700 are subsequently placed in fixture.Fixture may include frame for Primary containers 702 support sealing rings 500.It is that the operation of fixture can be automation or can manually perform.Alternatively, fixture can be formed Sealing between float 704 and Primary containers 702, without including sealing ring 500.Alternatively, such as pass through supersonic welding It connects；Or by applying heat or temperature gradient so that Primary containers 702 and float 704 are deformed and/or melted, sealing can be floating It is formed between son 704 and Primary containers 702.For convenience, this method for the sealing between float and Primary containers into Row description, but method described below is not directed in its application and is so limited, and may not need sealing and hold Row.
When the operation of fixture be automation when, engine promote collet include collet refer to or pressure member translation, with promote Make the compression that collet refers to.Engine can be attached to collet or pressure by handle such as cam handle and one or more gears Component.Substrate engagement and receiving object.When collet passes through engine driving, pressure member is remain stationary.When pressure member is logical When crossing engine driving, collet is remain stationary.Fixture may include disconnector, refer to promote pressure member to skid off collet 904, to remove clamping force.
Alternatively, fixture can be but not limited to collet fixture, o-ring, pipe clamp, hose clamp, spring perch, skin Carrying jig or tie such as band.Fixture may not need sealing ring and use, to provide the sealing between float and pipe.
As shown in FIG. 10A, blood plasma 801 can for example by liquid relief, aspirate, topple over etc. and taken from Primary containers 702 Out.Fig. 6 is returned to, in block 608, cleaning fluid can be added in Primary containers together with collector-shelter system.Figure 10B-10C shows the cleaning fluid 1002 being added in Primary containers 702, has close greater than at least buffy coat 802 Degree (that is, for example, can have greater than buffy coat but be less than the density of red blood cell, or can have greater than buffy The density of layer and both red blood cells).Alternatively, blood plasma 801 can be retained in Primary containers 702.When blood plasma 801 is retained in When in Primary containers 702, the density of blood plasma 801 can be for example by Iodixanol or any appropriate substance change, to change grade Divide density, to serve as cleaning fluid.It therefore, can when blood plasma 801 is retained in Primary containers 702 and density changes Not need cleaning fluid.
As shown in fig. 10d, collector-shelter system 400 is then added in Primary containers 702.The of collector 200 Two ends 208 and the inner wall of Primary containers 702 form sealing 1008, with prevent fluid before centrifugation, during centrifugation and after centrifugation It is flowed around collector 200.Sealing 1008 can be formed between the second end 208 and the inner wall of Primary containers, with from Before the heart, centrifugation during and centrifugation after maintain fluid-tight sealing engagement, and inhibit suspension any part be located at master It wants between the inside of container and the main body 204 of collector 200, or in the inside of Primary containers and the main body 204 of collector 200 Between flow.Sealing can be formed by following: interference engagement, lubricating grease (such as vacuum grease), adhesive, epoxy Resin, ultrasonic bonding, clamping (such as with ring or fixture), fits in the interior of the second end 208 and Primary containers at hot adhesion Insertion piece between wall etc..Locking ring 1004 can be placed in the shoulder 216 of collector 200 and the open end of Primary containers 702 On portion 710, to inhibit collector 200 relative to the translation of Primary containers 702.When being inserted into collector-shelter system 400, The a part of of cleaning fluid 1002 in Primary containers 702 can be by 214 discharge of intubation, and is stopped by shelter 402.Such as Along the line VI-VI obtain by the way that seen in the dotted line 406 in enlarged view 1006, the fluid of discharge can be flowed by window 218 Out and enter in Primary containers 702, although in the sealing being maintained between the second end 208 and the inner wall of Primary containers 702 Side.
In box 610, sequential density classification is executed.The snapshot of box 610 or sequential density classification step.Such as figure It is visible in 10E, in box 612, n-th of processing container including n displacement fluid is inserted into collector, so that n More than or equal to first (i.e. second, third, 4th etc.).The enlarged view of its cross section that VII-VII is obtained for along 1010 show cleaning fluid 1002 and erythrocyte sedimentation rate in the displacement fluid 312 and Primary containers 702 in processing container 302 Buffycoat 802.
Fig. 6 is returned to, in box 614, system is centrifuged to collect fraction or subfraction, and take out n-th of processing and hold Device.In block 616, operator determines whether to obtain required fraction or subfraction.When the fraction needed for obtaining or subfraction, Process can stop as shown in box 618, although process can continue until obtaining all fractions or subfraction.When not yet When fraction needed for obtaining or subfraction, process restarts at box 612.Processing container can also include processing solution, To realize the change to respective subfraction.Depending on the fraction or subfraction number for needing to separate and collect, it can be used two kinds Or more processing container and respective displacement fluid.Every kind of successive displacement fluid is bigger than previous displacement fluid density.It is similar Ground, each fraction or subfraction in succession are bigger than previous fraction or subfraction density.Once collecting, so that it may which analysis is continuous sub- Fraction, for example, for diagnosing, prognosis, research purpose, to measure component characteristics (i.e. complete blood count), these features are such as What is with time change etc..
Figure 10 F shows the collector-processing containment system 300 and Primary containers 702 of experience centrifugation.Along it is The enlarged view 1012 for the viewgraph of cross-section that VIII-VIII is obtained is shown between Primary containers 702 and processing container 302 The snapshot of fluid communication.As the cleaning fluid 1002 with the density greater than buffy coat 802 is in Primary containers 702 It moves down, buffy coat 802 is removed from float 704.With the first subfraction having greater than buffy coat 802 1014 but be less than cleaning fluid 1002 and buffy coat 802 remainder density displacement fluid 312 from processing container 302 flow in Primary containers 702, and the first subfraction 1014 moves upwardly through funnel 222 and hidden in Primary containers 702 Fluffy 214, and enter in processing container 302.As in Figure 10 G as it can be seen that the first subfraction 1014 and is set in processing container 302 Change of current body 312 and cleaning fluid 1002 are in Primary containers 702.
Processing container 302 including the first subfraction 1014 can then take out from collector 200, to undergo into one Step processing, analysis, storage etc..After taking out and processing container 302, processing solution can be added, although processing solution can be It is already present on before target material recycling in processing container.Processing container can for example be vibrated by turbine mixer. Before oscillation in liquid form, can dissolve shell or the processing solution (not shown) that is added in frangible shell, then may be used To be mixed with buffy coat, to realize conversion and form buffy coat-processing solution mixture.Buffy coat-processing Solution mixture can be then assigned on matrix such as microscopic slide.
Following process container and displacement fluid can be used for collecting the other subfraction of buffy coat 802, until all Subfraction is collected or until required subfraction is collected.Although sequential density classification is described as being executed with float and sealing ring, But sequential density classification may not need float, sealing ring or both and execute.It is hereafter for executing showing for sequential density classification Example method:
1. blood and float are added in pipe.
2. being centrifuged separated (i.e. blood plasma, buffy coat and the red blood cell) based on density to realize blood.
3. sealing ring is applied to around pipe and float at the bottom end of float；It clamps.
4. taking out blood plasma.
5. the cleaning fluid that the density that it has is greater than target density is added.
6. being inserted into collector-processing containment system, the first processing container includes the first displacement stream with the first density Body.
7. being centrifuged again.
It at present include the of buffy coat more smaller than the first displacement fluid density 8. taking out the first processing container One subfraction.
9. the second processing container includes second with the second density by the second processing container insertion collector Displacement fluid, second density are greater than the first displacement fluid and are less than cleaning fluid.
10. being centrifuged again.
It at present include smaller than the second displacement fluid density and the first displacement fluid of ratio 11. taking out the second processing container With the second subfraction of both the first subfractions bigger buffy coat of density.
12. step 9-11 is repeated using the bigger displacement fluid of successive density, to collect the bigger substate of successive density Point, until obtaining all required subfractions.
Any analysis method appropriate can be used in target material or technology is analyzed, although more specifically, extracellularly It include that intracellular protein marks with intracellular analysis；Color staining；Analysis of molecules；Genome analysis or foranalysis of nucleic acids, including but It is not limited to gene order-checking, DNA array, expression array, protein array and DNA hybridization array；(" ISH "-is used in situ hybridization In the tool of analysis DNA and/or RNA such as copy number changes)；Polymerase chain reaction (" PCR ")；Reverse transcription PCR；Or Branched DNA (tool that " bDNA "-is used to analyze DNA and/or RNA such as mRNA expression) analysis.These technologies can be with The target material for needing before analysis fixes, permeabilization processing and separation.One in intracellular protein that can be labeled It a bit include but is not limited to cytokeratin (" CK "), actin, Arp2/3, coronin, dystrophin, FtsZ, flesh Globulin, spectrin, tubulin, collagen, cathepsin D, ALDH, PBGD, Akt1, Akt2, c-myc, Guang day albumen Enzyme, survivin, p27kip, FOXC2, BRAF, phosphoric acid-Akt1 and 2, phosphoric acid-Erk1/2, Erk1/2, P38MAPK, waveform egg White, ER, PgR, PI3K, pFAK, KRAS, ALKH1, Twist1, Snail1, ZEB1, fibronectin, Slug, Ki-67, M30, MAGEA3, phosphate acceptor kinases, modified histone, chromatin-associated protein and MAGE.In order to fixation, it is permeabilization processing or Label, can be used fixative (such as formaldehyde, formalin, methanol, acetone, paraformaldehyde or glutaraldehyde), detergent (example Such as saponin(e, polyoxyethylene, digitonin, octyl β-glucoside, octyl β-glucosinolate, the thio pyrrole of 1-S- octyl β-D- It mutters glucoside, polysorbate20, CHAPS, CHAPSO, (1,1,3,3- tetramethyl butyl) phenyl-polyethylene glycol or octyl Phenol ethylene oxide) or marking agent (such as fluorescent labeled antibody, enzyme conjugation of antibodies, Pap stain, giemsa's stain or hematoxylin With Yihong stain).
After collection, target material can also be made to be imaged.In order to be imaged, the solution containing fluorescence probe can be used for marking Target material, to provide the fluorescence signal for for example identifying and characterizing by imaging.Before container is added in suspension, In Suspension is added after container but has undergone after centrifugation before centrifugation, or in suspension, and the solution containing fluorescence probe can To be added in suspension.Fluorescence probe includes the fluorescent molecule with ligand binding.Target material can have many different types Surface marker.Every class surface marker is the molecule that can be attached particular ligand such as antibody, such as antigen.Therefore, lead to The ligand and specific fluorescent molecular conjugate for making to be attached to particular surface marker are crossed, ligand can be used for classifying target material and survey Determine the specific type of target material present in suspension.The example of suitable fluorescent molecules includes but is not limited to quantum dot；It is commercially available can The dyestuff obtained, such as fluorescein, Hoechst, FITC (" fluorescein isothiocynate "), R- phycoerythrin (" PE "), Texas Red, allophycocyanin, Cy5, Cy7, cascade blue, DAPI (" 4', 6- diamidino -2-phenylindone ") and TRITC (" tetramethyl sieve Red bright isothiocyanates ")；Dye combinations such as CY5PE, CY7APC and CY7PE；And synthetic molecules such as self-assembly nucleic acid Structure.Many solution can be used, so that every kind of solution includes the different types of fluorescent molecule in conjunction with different ligands.
When collecting target material and when mixing in non-target material, the density of target material or non-target material can increase (such as By the way that weight (weight) is attached to target material or non-target material, or by adsorbing or taking the photograph with target material or non-target material Enter weight), or can reduce (such as by the way that buoy is attached to target material or non-target material, or by with target material or non- Target material absorption or intake buoy).Weight or buoy can be with ligand bindings.Target material can have many different types of Surface marker.Every class surface marker is the molecule that can be attached particular ligand such as antibody, such as antigen.Therefore, ligand It can choose and be attached to target material or non-target material for specificity.The example of approrpiate wts and/or buoy include but is not limited to by The pearl of metal, glass, ceramics, plastics or combinations thereof.After collection step and density change the step, second can be executed Sequential density classification is taken turns, to obtain the various components of purer target material or target material.
Aforementioned specification for illustration purposes uses specific nomenclature, to provide the thorough understanding of disclosure.So And it will be apparent to one skilled in the art that the details of concrete practice system and method described herein is not required 's.The foregoing description of specific embodiment is presented as an example, for the purpose of illustration and description.They are not intended to It is thoroughly describing or present disclosure is made to be limited to the precise forms for the precise forms.In view of instructing above, perhaps More modifications and variation are possible.Embodiment is shown and describes, so as to the principle and practice of best illustration present disclosure Using having and be such as suitable for considering so that those skilled in the art be allowed to optimise present disclosure and each embodiment Special-purpose various modifications.It is expected that scope of the present disclosure limited by the appended claims and its equivalent It is fixed.

Claims (21)

1. a kind of method for collecting target material from sample, the method includes the following steps:
It is centrifuged the Primary containers containing the sample, to realize the separating based on density of blood；
It will be in open end of the collector insertion containing the Primary containers of the sample；
N-th of processing container is inserted into it is intracavitary in the collector,
N displacement fluid is added in n-th of the processing container, the n displacement fluid, which has, is greater than the sample The density of the n subfraction of fraction, the n subfraction include at least part of the target material；With
Make the Primary containers, the collector and n-th of processing container centrifugation, the n displacement fluid is via institute The intubation for stating collector is flow in the Primary containers, by the n subfraction via the collector intubation from In the Primary containers displacement to n-th of the processing container,
Wherein the sample fraction is a part of the sample, and
Wherein the subfraction of the fraction is a part in a part of the sample.
2. method described in claim 1, wherein repeat by n-th of processing container insertion, will be described in the addition of n displacement fluid The step of n-th of processing container neutralization is centrifuged the Primary containers, the collector and n-th of processing container, until All required subfractions are obtained from the sample, wherein every kind of successive displacement fluid, which has, is greater than every kind of previous displacement fluid Density.
3. method as claimed in claim 2, wherein n is greater than or equal to 2.
4. method as claimed in claim 2, wherein at least one of described subfraction includes the target material.
5. method as claimed in claim 3, wherein at least one of described subfraction does not include the target material.
6. method as claimed in claim 2, wherein the fraction is buffy coat, and wherein the subfraction is the blood The different piece of heavy buffycoat.
7. method as claimed in claim 2, wherein the displacement fluid is selected from: being coated with the colloidal silica of polyvinylpyrrolidone Solution, polysaccharide solution, organic solvent, liquid wax, gas, ionic liquid, the solution based on polymer, surface-active of silicon particle Agent, hydrofluorocarbon, perfluocarbon, the liquid based on silicon and combinations thereof.
8. method as claimed in claim 2, wherein the displacement fluid none mixed any one of with other displacement fluids.
9. method described in claim 1, wherein the n subfraction includes the target material.
10. method described in claim 1 further comprises that float is added in the Primary containers before centrifugation.
11. method described in any one of claim 10 further comprises by being clamped in the float and Primary containers after centrifugation Between formed sealing.
12. method described in claim 1, wherein the target material include erythroblast, leucocyte, circulating tumor cell, Conveyor screw, malaria inducer or immunocyte.
13. method described in claim 1 further comprises the step of being after collection imaged the target material.
14. method described in claim 1, wherein the target material undergoes analysis of molecules after collection.
15. method described in claim 1, wherein at least one analyte is separated from the target material.
16. method as claimed in claim 2, wherein the displacement fluid is oil.
17. method described in claim 16, wherein the oil is selected from olive oil, mineral oil, silicone oil, immersion oil, paraffin oil And silicone oil.
18. method of claim 7, wherein the organic solvent is selected from Iodixanol, Isosorbide-5-Nitrae-dioxanes, acetonitrile, acetic acid It is ethyl ester, the tert-butyl alcohol, cyclohexanone, methylene chloride, tert-pentyl alcohol, methyl tertiary butyl ether(MTBE), butyl acetate, hexanol, nitrobenzene, toluene, pungent Alcohol, octane, propylene glycol carbonate and sulfolane.
19. method of claim 7, wherein the perfluocarbon is selected from perfluorodecalin, perfluor perhydro phenanthrene, perfluorooctane base Bromide, perfluor ketone, perfluorocyclopentanone, perfluoropolyether and perfluorocyclohexanone.
20. method as claimed in claim 2, wherein the displacement fluid is selected from fluorinated ketone and hydrofluoroether.
21. method of claim 7, wherein the liquid based on silicon is selected from fluorosilicone, siloxanes and benzyl silicon Oxygen alkane.
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