EP1105711A2 - Extraction fluidique de prelevements ayant fait l'objet d'une microdissection - Google Patents

Extraction fluidique de prelevements ayant fait l'objet d'une microdissection

Info

Publication number
EP1105711A2
EP1105711A2 EP99935859A EP99935859A EP1105711A2 EP 1105711 A2 EP1105711 A2 EP 1105711A2 EP 99935859 A EP99935859 A EP 99935859A EP 99935859 A EP99935859 A EP 99935859A EP 1105711 A2 EP1105711 A2 EP 1105711A2
Authority
EP
European Patent Office
Prior art keywords
capillary
reaction chamber
sample
chamber
laminate layer
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP99935859A
Other languages
German (de)
English (en)
Inventor
Thomas M. Baer
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Molecular Devices LLC
Original Assignee
Arcturus Engineering Inc
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 US09/357,423 external-priority patent/US7473401B1/en
Application filed by Arcturus Engineering Inc filed Critical Arcturus Engineering Inc
Publication of EP1105711A2 publication Critical patent/EP1105711A2/fr
Withdrawn legal-status Critical Current

Links

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
    • B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
    • B01L3/5082—Test tubes per se
    • B01L3/50825—Closing or opening means, corks, bungs
    • 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
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502707—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the manufacture of the container or its components
    • 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
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502746—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the means for controlling flow resistance, e.g. flow controllers, baffles
    • 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
    • B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
    • B01L3/5082—Test tubes per se
    • 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
    • G01N1/2813—Producing thin layers of samples on a substrate, e.g. smearing, spinning-on
    • 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
    • B01L2300/04—Closures and closing means
    • B01L2300/046—Function or devices integrated in the closure
    • 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
    • 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
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0825—Test strips
    • 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
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0848—Specific forms of parts of containers
    • B01L2300/0858—Side 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
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/0867—Multiple inlets and one sample wells, e.g. mixing, dilution
    • 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
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/0883—Serpentine channels
    • 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
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0887—Laminated structure
    • 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
    • B01L2400/0406—Moving fluids with specific forces or mechanical means specific forces capillary forces
    • 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
    • B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
    • B01L2400/0481—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure squeezing of channels or chambers
    • 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
    • B01L2400/06—Valves, specific forms thereof
    • B01L2400/0688—Valves, specific forms thereof surface tension valves, capillary stop, capillary break
    • 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
    • B01L9/00—Supporting devices; Holding devices
    • B01L9/52—Supports specially adapted for flat sample carriers, e.g. for plates, slides, chips
    • B01L9/527—Supports specially adapted for flat sample carriers, e.g. for plates, slides, chips for microfluidic devices, e.g. used for lab-on-a-chip
    • 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
    • G01N1/2813—Producing thin layers of samples on a substrate, e.g. smearing, spinning-on
    • G01N2001/2833—Collecting samples on a sticky, tacky, adhesive surface
    • G01N2001/284—Collecting samples on a sticky, tacky, adhesive surface using local activation of adhesive, i.e. Laser Capture Microdissection

Definitions

  • the invention relates generally to the liquid extraction of microdissected samples. More particularly, the invention relates to the liquid extraction of microdissected tissue samples through fluidic circuits, including the interrelationships between microdissection sample carriers and microdissection analysis vessels.
  • microdissection techniques and processing are known to those skilled in the art. For example, a conventional microdissection is typically is typically performed with small surgical instruments.
  • a problem with this technology has been that subsequent processing of the microdissected sample is difficult because of the small size of the sample. Therefore, what is required is solution that facilitates processing of microdissected samples.
  • Another problem with this technology has been that use of a relatively large amount of reaction buffer and/or subsequent reagents, which dilutes the sample constituents, can make obtaining data from the comparatively small sample difficult. Therefore, what is also required is a solution that uses a smaller volume of reaction buffer and/or other reagents.
  • One approach, in an attempt to solve the above-discussed problems involves using a carrier film to capture and transport the microdissected sample. This film and sample are then both dropped into the centrifuge tube where the sample is contacted by the reaction buffer.
  • a goal of the invention is to simultaneously satisfy the above-discussed requirements of facilitating and simplifying subsequent processing, reducing the volume of reagents, and economy which, in the case of the prior art, are mutually contradicting and are not simultaneously satisfied.
  • One embodiment of the invention is based on a biological sample processing system, comprising: a laminated film sample processing device including a reaction chamber mated with a biological sample carrier to form a fluidic circuit.
  • a fluidic circuit comprising: a reaction chamber; a sample carrier mating surface coupled to said reaction chamber; and a conduit coupled to said reaction chamber.
  • Another embodiment of the invention is based on a method of processing a biological sample, comprising: providing a sample carrier with a biological sample; and mating said sample carrier to a laminated film sample processing device having a reaction chamber to form a fluidic circuit, wherein said biological sample is positioned within said reaction chamber.
  • Another embodiment of the invention is based on a microdissected sample extraction device, comprising: a fill port defined at least in part by a middle laminate layer and a bottom laminate layer; a fill port-to-reaction chamber capillary coupled to said fill port, said fill port-to-reaction chamber capillary defined at least in part by said middle laminate layer, said bottom laminate layer and a top laminate layer, said fill port-to-reaction chamber capillary defining a middle stop junction that extends through said top laminate layer; a spacer coupled to said top laminate layer, said spacer including a microdissected sample film carrier mating surface; an reaction chamber coupled to said fill port-to-reaction chamber capillary through said middle stop junction, said reaction chamber defined at least in part by said top laminate layer and said spacer; and an reaction chamber exit capillary coupled to said reaction chamber, said reaction chamber exit capillary defined at least in part by said middle laminate layer, said bottom laminate layer and said top laminate layer, said extraction chamber exit capillary defining a second stop junction that extend
  • Another embodiment of the invention is based on an apparatus, comprising: a multiple step fluidic device for laser capture microdissection, said multiple step fluidic device including a transfer film containing the sample to be analyzed and a surface that is spaced apart from said transfer film so as to define a fluid volume, said surface being connected to an exit stop junction that functions as an exit port for a reaction buffer.
  • Another embodiment of the invention is based on a method, comprising: providing a multiple step fluidic device for laser capture microdissection, said multiple step fluidic device including i) a transfer film to which a portion of a sample is adhered and ii) a surface that is spaced apart from said transfer film so as to define a fluid volume, said surface being connected to an exit stop junction that functions as an exit port for a reaction buffer; contacting said portion with said reaction buffer; and then removing said reaction buffer from said fluid volume.
  • FIG. 1 illustrates a side schematic view of a microcentrifuge tube and cap, representing an embodiment of the invention.
  • FIG. 2 illustrates a side schematic view of the microcentrifuge tube and cap of FIG. 1 with the cap inserted into the tube, representing an embodiment of the invention.
  • FIG. 3 illustrates a side schematic view of the microcentrifuge tube and cap of FIGS. 1 and 2 after spinning, representing an embodiment of the invention.
  • FIG. 4 illustrates a side schematic view of a microcentrifuge tube and cap, representing an embodiment of the invention incorporating a smaller transfer film area.
  • FIG. 5 illustrates a side schematic view of the microcentrifuge tube and cap of FIG. 4 with the cap inserted into the tube, representing an embodiment of the invention.
  • FIG. 6 illustrates a side schematic view of the microcentrifuge tube and cap of FIGS. 4 and 5 after spinning, representing an embodiment of the invention.
  • FIG. 7 illustrates a top schematic view of an insert or plug incorporating a stop junction, representing an embodiment of the invention.
  • FIG. 8 illustrates a side schematic view of another insert or plug, representing an embodiment of the invention.
  • FIG. 9 illustrates a top schematic view of a laser capture microdissection film carrier, representing an embodiment of the invention.
  • FIG. 10 illustrates a side schematic view of a laminated film device mated with the film of a laser capture microdissection film carrier to form a fluidic circuit, representing an embodiment of the invention.
  • FIG. 11 illustrates a top schematic view of the fluidic circuit shown in FIG. 10, representing an embodiment of the invention.
  • FIG. 12 illustrates a side schematic view of a cap sealed to a laminated assembly, representing an embodiment of the invention.
  • FIG. 13 illustrates a side schematic view of a laminated extraction capillary/stop junction assembly, representing an embodiment of the invention.
  • FIG. 14 illustrates a side schematic view of the cap/laminate of FIG. 12 inserted into a microcentrifuge tube, representing an embodiment of the invention.
  • FIG. 15A illustrates a top schematic view of the bottom laminate of an extraction device, representing an embodiment of the invention.
  • FIG. 15B illustrates a top schematic view of the middle laminate of an extraction device, representing an embodiment of the invention.
  • FIG. 15C illustrates a top schematic view of the top laminate of an extraction device, representing an embodiment of the invention.
  • FIG. 15D illustrates a side schematic view of the laminates depicted in FIGS 15A-15C together with a microdissected sample carrier, representing an embodiment of the invention.
  • FIG. 16 illustrates a top schematic view of a bottom laminate, representing an embodiment of the invention.
  • FIG. 17 illustrates a top schematic view of a top laminate, representing an embodiment of the invention.
  • FIG. 18 illustrates a top schematic view of a middle laminate before cutting, representing an embodiment of the invention.
  • FIG. 19 illustrates a schematic view of a laser cutting pattern for the middle laminate depicted in FIG. 18, representing an embodiment of the invention.
  • FIG. 20 illustrates a top schematic view of a middle laminate after cutting, representing an embodiment of the invention.
  • FIG. 21 A illustrates a top schematic view of the bottom laminate of a single stage microdissected sample extraction device, representing an embodiment of the invention.
  • FIG. 21B illustrates a top schematic view of the middle laminate of a single stage microdissected sample extraction device, representing an embodiment of the invention.
  • FIG. 21C illustrates a top schematic view of the top laminate of a single stage microdissected sample extraction device, representing an embodiment of the invention.
  • FIG. 21D illustrates a top schematic view of the spacer of a single stage microdissected sample extraction device, representing an embodiment of the invention.
  • FIG. 21E illustrates a top schematic view of the assembled single stage microdissected sample extraction device, representing an embodiment of the invention.
  • FIG. 22 illustrates a top detail view of the microdissected sample extraction device depicted in FIG 2 IE, representing an embodiment of the invention.
  • FIG. 23 A illustrates a top schematic view of the bottom laminate of a two stage microdissected sample extraction device, representing an embodiment of the invention.
  • FIG. 23B illustrates a top schematic view of the middle laminate of a two stage microdissected sample extraction device, representing an embodiment of the invention.
  • FIG. 23 C illustrates a top schematic view of the top laminate of a two stage microdissected sample extraction device together with a foam ring and coversheet with hole for pumping sample and dilutent, representing an embodiment of the invention.
  • FIG. 23D illustrates a top schematic view of the extraction chamber defining spacer of the two stage microdissected sample extraction device together with a release layer, representing an embodiment of the invention.
  • FIG. 23E illustrates a top schematic view of a dilution chamber defining spacer of the two stage microdissected sample extraction device, representing an embodiment of the invention.
  • FIG. 23F illustrates a top schematic view of a cover for the extraction chamber defining spacer of the two stage microdissected sample extraction device, representing an embodiment of the invention.
  • FIG. 23G illustrates a top detail view of the assembled two stage microdissected sample extraction device, representing an embodiment of the invention.
  • the invention includes any laminated film device mated with any microdissected sample carrier to form a fluidic circuit.
  • the laminated film device can be termed an extraction device.
  • the microdissected samples can be obtained in any manner including, for example, laser capture microdissection, laser pressure catapulting, laser trapping, laser cutting and/or ablation, mechanical cutting, etceteras.
  • the invention can include an extraction chamber.
  • the extraction chamber can be defined in part by the sample carrier.
  • the extraction chamber can also be defined in part by a spacer ring.
  • the invention can include one or more capillaries.
  • the capillaries are pipes or conduits that permit mass transport within the extraction device.
  • An end of a capillary where fluid flows and then stops can be termed a stop junction.
  • a capillary end where fluid is introduced can be termed a fluid well or fluid port.
  • the capillaries can couple structure features located within, or outside, the extraction device.
  • the stop junction(s) and/or fill port(s) can be upgraded with the addition of pump.
  • Such a pump can be a simple externally actuated bubble (aka blister) formed in one, or more, of the laminate layers.
  • An intervening resilient layer e.g., foam
  • the pump blister can have a hole that can be covered by the operator's finger so that when covered and the pump blister is depressed the increase in air pressure in the fluidic circuit causes fluid to move in the circuit.
  • the pump blister hole can also act as a fluid well or fluid port.
  • Mass i.e., fluid, solid and gas
  • capillary force(s) e.g., pumping force(s)
  • acceleration force(s) e.g., centripetal acceleration from a laboratory centrifuge into which the entire extraction device can be placed.
  • a pump or acceleration force is usually required to fill and/or empty a chamber.
  • Extraction devices that are shaped to fit at least partially into centrifuge tubes can be termed darts, which is descriptive of their shape.
  • the flow rate of fluid within the capillary can be controlled by the diameter of the capillary.
  • the volumetric flow rate within the capillaries can be a function of the hydrostatic pressure. Thus, a large volume of fluid at a well generating a large head pressure will tend to result in a higher flow rate.
  • the ratio of a capillary inlet diameter to a capillary outlet diameter can be used to control the volumetric flow. For instance, a small capillary pulling from large well will have a higher volumetric flow than the same capillary pulling from smaller well.
  • Capillary forces can also be used to move the fluid through the device. By manufacturing the device out of hydrophilic materials the water based fluid will be drawn into the capillaries by capillary action.
  • the fluid will move until it reaches an exit port with a small diameter, i.e. a stop junction.
  • the fluid can be forced through the stop junction by increasing the forces on the fluid, for example by using centrifugal acceleration or increased air pressure.
  • the invention can include a dilution chamber defined by the laminated film device. A dilutent can be added to the device after the digestion reagents and the device can be placed in a centrifuge to move the dilutent plus the digested sample into a dilution chamber.
  • the invention can also include reagents deposited in the chamber(s), conduit(s), well(s) and/or stop junction(s) to change the surface tension or hydrophilicity of the laminate material, or even the fluid.
  • the deposited reagents can also include digestion compounds, analysis reagents such as antibodies or nucleic acid probes.
  • the context of the invention is microdissected sample analysis, especially cellular tissue analysis.
  • the extraction device can be coupled to other analysis equipment such as a filter, a hybridization chamber, a PCR chamber, assay equipment, etceteras.
  • the particular manufacturing process used for fabricating the laminated extraction devices should be inexpensive and reproducible.
  • the laminate layers can be processed by standard laminated film converting process incorporating mechanical punching or cutting of continuous rules of laminated films and assembly onto reels. This process is well known to those skilled in the art and is called a web based process.
  • the devices can also be manufactured by a combination of mechanical and laser cutting (e.g., 25 W CO2 laser).
  • the laminate layers can be joined by a continuous roll calendering or adhesive process.
  • the laminate layers can also be joined (or additional structural components, such as covers, added) by ultrasonic welding and/or heat staking.
  • the particular manufacturing process used for fabricating the laminated extraction devices is not essential to the invention as long as it provides the described functionality. Normally those who make or use the invention will select the manufacturing process based upon tooling and energy requirements, the expected application requirements of the final product, and the demands of the overall manufacturing process.
  • the particular material used for laminated extraction devices should be biologically and chemically inert. It is preferred that the laminate materials be a hydrophilic polymer. For the manufacturing operation, it is an advantage to employ a polyester material. Selected areas of the materials can have surface treatment to direct and control the flow of fluid such as texturing and/or plasma or chemical treatment. These treatments can vary the surface tension to make the materials more wettable.
  • a purpose of the invention is to provide a method for extracting cellular material from a laser capture microdissection (LCM) film that might employ a variety of different geometries, and require a small volume of reaction buffer.
  • Current techniques require inserting the LCM film carrier into the fluid. After the extraction reaction the LCM film carrier is removed from the reaction buffer and the liquid reaction buffer is then processed in subsequent stages.
  • One method to achieve this goal is to incorporate a simple stop junction into the design of a microcentrifuge tube as illustrated in FIGS. 1-8.
  • This stop junction can consist of a small hole 10 or multiple holes in an insert in the tube that prevents the fluid from passing through the hole unless some force, say a centrifugal force, is applied to the liquid.
  • the force required to cause the fluid to pass through the hold can be adjusted by varying the size of the hole.
  • the processing steps include (a) applying the buffer to the top of the insert 100, as shown in FIGS 1 and 4.
  • the buffer is prevented from penetrating the insert 100 by the stop junction forces.
  • the fluid, and therefore the size of the vessel is adjusted so as to prevent forcing the fluid completely through the hole when the film carrier inserted into the tube, as shown in FIGS. 2 and 5. Extraction takes place with the apparatus in the configuration illustrated in FIGS. 2 and 5.
  • the reaction vessel assembly is placed in a centrifuge (c) and spun at a velocity to supply sufficient force to move the liquid past the stop junction and into the reservoir, as shown in FIGS. 3 and 6.
  • the film carrier is then removed without disturbing the fluid contents.
  • the fluid can be removed from the reagent vessel using a thin pipette or a syringe. Inserts or plugs, as shown in FIGS. 7-8, can be utilized inside the vessel.
  • a purpose of the invention is to provide a LCM film carrier that has a large surface area in order to cover a large portion of the tissue sample and yet require a small liquid volume to digest the transferred tissue. This can be accomplished by providing a laser capture microdissection (LCM) film carrier as part of a capillary assembly.
  • LCM laser capture microdissection
  • the invention incorporates an LCM film carrier 900, as shown in FIG. 9, into a fluidic assembly that allows a thin layer of the liquid to contact the film.
  • the film 910 can be spaced off from a mating surface by a precision spacer that can include of a piece of double sided adhesive 920 of an appropriate thickness, for example, approximately 100 microns.
  • This tape creates a gap between the film carrier and the mating surface as indicated in FIG. 10 and seals the liquid in to the interior area of the film carrier.
  • An appropriate volume of liquid reagents are applied to the mating surface prior to sealing with the film carrier. Referring to FIG. 11 , the liquid is extracted from the sealed assembly through an exit port 930 by applying air pressure to the vent hole 940.
  • the assembly can be inserted in a centrifuge and with the exit port at a larger radius then the vent hole and the exit port attached to a suitable reagent vessel.
  • This assembly can be rotated at sufficient angular velocity to overcome the stop junction forces and empty the fluid through the exit port and into the reagent vessel.
  • FIGS. 12-14 the alternative embodiment is shown embodied in combination with a microcentrifuge tube assembly.
  • the cap 1200 that carries the LCM transfer film is spaced away from the mating surface with a double sided adhesive spacer so as to define a fluid volume.
  • 1210 In FIG. 13 the vent hole and the exit stop junction 1220 of the mating surface can be seen.
  • Multiple vent holes can be used to allow application port and air vents so that the reagents can be applied through the application port after assembly of the device. Liquid can be loaded onto the center of the laminate assembly and then the cap can be placed on top.
  • the liquid volume can be metered so as to fill the reaction region and wet the surface of the cap (i.e., the LCM transfer film and acquired portion of sample) but not be forced out through stop junction.
  • Cap/laminate assembly 1230 is then inserted into a microcentrifuge tube. After reaction, the microcentrifuge tube assembly can be spun at a sufficient rotational velocity to allow fluid to pass through stop junction and rest in the bottom of tube. This technique will also work with microtiter plates.
  • the film carrier has dimensions of 1 cm. by 1 cm. and that the double sided tape thickness is 100 microns.
  • the resulting enclosed volume will be only 10 mm 3 or 10 microliters.
  • the forces exerted on the enclosed liquid can be sufficient to cause the extraction product to pass through a stop junction that is contiguous with the enclosed volume and be collected in the bottom of a tube.
  • Example 2 Referring to FIGS. 15A-15D, a single stage extraction device with a pump is depicted.
  • this single stage extraction device includes a base laminate 1510.
  • the base laminate 1510 includes an exit port 1520.
  • this single stage extraction device includes a middle laminate 1530.
  • the middle laminate 1530 includes a first orifice defining a pump area 1540.
  • the middle laminate 1530 includes a second orifice defining a reaction area 1550.
  • the reaction area 1550 can correspond to a three- dimensional extraction chamber.
  • the pump area 1540 is connected to the reaction area 1550 via a first capillary 1560.
  • a second capillary 1570 is also connected to the reaction area 1550.
  • the middle laminate area 1530 can be made of a sheet of polymer having a first sticky side and a second sticky side, thereby defining a double adhesive layer.
  • this single stage extraction device includes a top laminate 1580.
  • the top laminate 1580 includes a first orifice 1590 that is coincident with the reaction area 1550. Together, the second orifice of the middle laminate 1530 and the first orifice of the top laminate 1580 cooperate to define a extraction chamber for extraction of components from the sample.
  • the top laminate 1580 includes a bi-position pump blister 1595 that is coincident with the pump area 1540.
  • the base laminate 1510, the middle laminate 1530, and the top laminate 1580 can be seen joined together to form the single stage extraction device.
  • a film carrier 1505 is depicted adjacent the top laminate 1580. Together, the bottom of the film carrier 1505, the first orifice 1590 and the reaction area 1550 cooperate to define the extraction chamber.
  • the pump area 1540 can be provided with a reaction buffer (aka extraction fluid).
  • a microdissected sample on the film carrier 1505 is then introduced, and the extraction chamber closed, by placing the film carrier 1505 on the top surface of the top laminate 1580.
  • the bi-position pump blister 1595 is then actuated to force reaction buffer into the extraction chamber so that it contacts the microdissected sample.
  • the bi-position pump blister 1595 can be further actuated to force extraction fluid that is carrying aspects of the sample toward the exit port 1520.
  • Example 3 Referring now to FIGS. 16-22, another single stage extraction device and a method for manufacture thereof will now be described. The device will be described first, then the component parts of the device will be described, then the process of making the device will be described, and then the process of operating the device will be described.
  • the assembled single stage extraction device is depicted.
  • This device includes a spacer 2170 that defines in-part an extraction chamber 2180. More generically, the extraction chamber 2180 can be termed a reaction chamber.
  • the extraction chamber 2180 is coupled to first capillary 2130.
  • the first capillary 2130 is coupled to a fill port 2110.
  • the extraction chamber 2180 is also coupled to a second capillary 2140. It can be appreciated that the spacer 2170 overlies and is aligned with the two capillary stop junction holes 2160. Thus, the interior of the spacer 2170 defines the extraction chamber
  • the spacer 2170 includes a mating surface 2190.
  • the mating surface 2190 is for attachment (mating) to a biological sample carrier (not shown in FIG. 2 IE), for example, a laser capture microdissection transfer film carrier.
  • FIGS. 21 A-21E the component parts of the single stage extraction device are depicted.
  • the bottom laminate reflects the outline of the device and includes no specific additional structural features.
  • the middle laminate layer 2120 includes a fill port 2110.
  • the fill port 2110 is connected to a first capillary 2130.
  • the middle laminate 2120 also includes a second exit capillary 2140.
  • the fill port 2110, the first capillary 2130, and the second capillary 2140 can all be seen in FIG. 18.
  • the top laminate 2150 of the single stage extraction device is depicted.
  • the top laminate 2150 includes a fill port hole 2155 and two capillary stop junction holes 2160.
  • the capillary stop junction holes 2160 in the top laminate 2150 align with the ends of the capillaries 2130 and 2140 depicted in FIG. 21B.
  • the fill port hole 2155 in FIG. 21C aligns with the fill port 2110 in FIG. 2 IB.
  • a spacer 2170 in the form of a ring is depicted.
  • the spacer 2170 includes a microdissected sample film carrier mating surface 2175.
  • a series of laminate stock strips are depicted.
  • a laminate stock strip for the bottom laminate is shown.
  • the bottom laminate includes four tooling pin holes 1610.
  • the bottom laminate should be a hydrophilic polymer, for example, a polyester with an optional surfactant treatment.
  • a suitable hydrophilic polymer are those manufactured to have anti-fog properties.
  • FIG. 17 a laminate stock strip for the top laminate is depicted.
  • the laminate stock strip for the top laminate includes four tooling pin holes 1710 and a number of stop junction holes 1720.
  • Each laminate stock strip includes two rows of single stage extraction devices (depicted in fig. 21).
  • Each of the single stage extraction devices has a top laminate portion with two stop junction holes 1720.
  • a variety of exemplary dimensions in inches are shown in FIG. 17.
  • the top laminate stock strip material should be a hydrophilic polymer, again for example, a polyester with optional surfactant treatment. Again it useful if the hydrophilic polymer has anti-fog properties.
  • the middle laminate includes four tooling pin holes 1810 and a number of other structural features.
  • the middle laminate often is chosen to have pressure sensitive adhesive on both surfaces.
  • a variety of exemplary dimensions in inches are shown in FIG. 18. Of course, the invention is not limited to any specific dimensions. It can be appreciated from FIG. 18 that the two rows of single stage extraction devices point toward one another on this stock strip.
  • the top, middle, and bottom films are assembled using the alignment holes 1610,1710, and 1810 to align the three layers. The assembly can be pressed together using a hand roller, or a rolling mill or other techniques known to those skilled in the art.
  • FIG. 19 a laser cutting track for the perforated laminate assembly is depicted.
  • This cutting track separates the double row of devices illustrated in figures 16, 17 and 18 into two separate single rows.
  • the devices or darts can then be individually separated using a simple cutting device such as a scissors.
  • a simple cutting device such as a scissors.
  • FIG. 19 a variety of exemplary dimensions are depicted in FIG. 19 and the invention is not limited to these dimensions.
  • the perforated laminate stock strip depicted in FIG. 18 is located with respect to the pin holes 1810 and processed by a carbon dioxide laser to form the cut lines 1910 that are depicted in FIG. 19.
  • FIG. 20 the laser cut laminate stock strip that results from processing the strip shown in FIG. 18 in accordance with the trace shown in FIG. 19 is shown.
  • the two parallel, facing rows of single stage extraction devices can be held together with tabs 2010 provided that the cutting trace is appropriately interrupted.
  • a transfer film (not shown) carrying a microdissected sample can be mated with the microdissected sample film carrier mating surface 2175, thereby completing the extraction chamber 2180.
  • An extraction fluid is applied to the fill port 2110 with sufficient volume to fill the extraction chamber and fill capillary 2130. Capillary forces draw the extraction fluid into the fill capillary 2130 and extraction chamber formed by ring 2170 and the transfer film. Stop junction forces prevent the fluid from exiting the extraction chamber.
  • the extraction fluid in the fill port 2110 will be driven through the first capillary 2130 to the extraction chamber 2180 whereupon it will react with (aka digest) the microdissected sample.
  • the extraction fluid that carries portions (or all) of the microdissected sample will pass from the extraction chamber 2180 into the second capillary 2140 and thence pass out of the single stage extraction device at a tip 2190.
  • the size of the first or entrance stop junction hole 1720 can be made slightly larger than the exit stop junction hole in order to provide greater stop junction forces at this junction, holding the extraction fluid in the extraction chamber until the centrifuge rpm is increased.
  • reaction buffer from fill port 21 10 will be contained by spacer 2170 and eventually pass through the second capillary 2140.
  • Example 4 Referring to FIGS. 23A-23G, a two stage extraction device is depicted.
  • the bottom laminate 2310 includes four capillary stop junction holes 2320. These stop junction holes can be of slightly different diameters thus requiring different pressures for the fluid to pass through the stop junction.
  • the middle laminate 2330 includes a fill port 2335.
  • a first capillary 2340 is coupled to the fill port 2335.
  • the middle laminate 2330 includes a second capillary 2345.
  • the middle layer 2330 also includes a third capillary 2350 which terminates at a tip 2355.
  • a top laminate 2360 includes a layer of foam 2362 defining a pump space 2364.
  • the top laminate 2360 includes a hole 2366.
  • a cover sheet 2368 is placed over the layer of foam 2362.
  • a spacer 2370 in the form of a ring is shown coupled to a release layer 2372.
  • the ring can be a piece of plastic with adhesive on both sides.
  • the release layer 2372 can be a piece of silicone coated paper.
  • a layer of foam 2380 with pressure sensitive adhesive on both sides has a hole 2382.
  • the hole 2380 will define a dilution chamber.
  • a cover layer 2390 is depicted. The cover layer 2390 is placed on top of foam 2380.
  • FIG. 23 G shows a number of exemplary dimensions associated with the two stage extraction device.
  • the invention is not limited to any particular dimensions.
  • a reaction buffer (aka extraction fluid) can be located in the fill port 2335 before shipment from the manufacturer or can be placed in the well by the end user.
  • the release layer 2372 is removed from the spacer 2370 and a sample film carrier (not shown) is mated with the microdissected sample film carrier mating surface of the spacer 2370 such that the microdissected sample is introduced into the extraction chamber.
  • the extraction buffer is then applied to the fill port 2335.
  • the hole in the cover sheet 2368 is then covered and the pump is actuated by compressing the foam 2362 to initiate pumping, thereby forcing reaction buffer in the fill port 2335 through the capillary 2340 and then into the extraction chamber.
  • dilutent is applied to the entrance port 2335.
  • the pump is be actuated by compression (i.e., depressing the cover 2362).
  • Reaction buffer , microdissected sample, and the dilutent will then be forced from the extraction chamber into the dilution chamber defined by hole 2382.
  • the microdissected sample can be processed by a first volume of reaction buffer that is then increased to a second volume by the addition of the dilution fluid.
  • This has significant advantages in that the very small microdissected sample can be processed by a correspondingly small amount of reaction buffer while subsequent processing can be carried out on a larger volume of material that includes the dilution fluid.
  • the dilution product can be forced through the third capillary 2350 toward the tip 2355.
  • Other reagents could be coated within the capillaries and/or stop junction holes.
  • a practical application of the invention that has value within the technological arts is the extraction of organic molecules from microdissected samples. Further, the invention is useful in conjunction with analyzing DNA (useful for the purpose of determining susceptibility to disease), or in conjunction with identifying malignancies (useful for the purpose of diagnosis), or the like. There are virtually innumerable uses for the invention, all of which need not be detailed here.
  • An extraction device representing an embodiment of the invention, can be cost effective and advantageous for at least the following reasons.
  • the invention permits small microdissected samples to be digested by small volumes of reagents.
  • the invention permits small digested volumes to be diluted to larger volumes.
  • the invention permits processing of microdissected sample in an economic manner.

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Analytical Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Hematology (AREA)
  • Clinical Laboratory Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Dispersion Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Automatic Analysis And Handling Materials Therefor (AREA)
  • Sampling And Sample Adjustment (AREA)

Abstract

Cette invention a trait à des dispositifs et aux techniques afférentes permettant le traitement d'échantillons de microdissection par capture laser (LCM). Ce système de traitement de prélèvement biologique comprend un dispositif de traitement de prélèvement à film laminé comportant une chambre à réaction connectée à un support de prélèvement biologique afin de constituer un circuit fluidique. Le dispositif fluidique à plusieurs niveaux comporte un film de transfert de LCM et une surface distante du film de transfert de manière à définir un volume fluidique. Il est possible d'enlever le tampon de réaction par un orifice d'évacuation ou un raccordement d'arrêt de sortie, au niveau de la surface. Entre autres avantages, ces dispositifs et techniques facilitent le traitement ultérieur du fait de la réduction de la quantité des réactifs et permettent de réduire les coûts. Le tampon de réaction peut, par exemple, être commodément retiré du film de transfert de LCM.
EP99935859A 1998-07-21 1999-07-21 Extraction fluidique de prelevements ayant fait l'objet d'une microdissection Withdrawn EP1105711A2 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
US9374498P 1998-07-21 1998-07-21
US93744P 1998-07-21
US09/357,423 US7473401B1 (en) 1997-12-04 1999-07-20 Fluidic extraction of microdissected samples
US357423 1999-07-20
PCT/US1999/016635 WO2000005587A2 (fr) 1998-07-21 1999-07-21 Extraction fluidique de prelevements ayant fait l'objet d'une microdissection

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EP1105711A2 true EP1105711A2 (fr) 2001-06-13

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EP (1) EP1105711A2 (fr)
JP (1) JP2002521668A (fr)
AU (1) AU5124499A (fr)
CA (1) CA2338246A1 (fr)
MX (1) MXPA01000691A (fr)
WO (1) WO2000005587A2 (fr)

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US7776273B2 (en) 2000-04-26 2010-08-17 Life Technologies Corporation Laser capture microdissection (LCM) extraction device and device carrier, and method for post-LCM fluid processing
EP1672349A3 (fr) * 2000-04-26 2014-02-19 Life Technologies Corporation Dispositif d'extraction à microdissection laser (LCM), support de dispositif et procédé de traitement par fluide post-LCM
US6790636B1 (en) 2000-06-14 2004-09-14 The United States Of America As Represented By The Department Of Health And Human Services Rapid fluorescent labeling of tissue for microdissection using fluorescent specific binding agents
DE10057292C2 (de) * 2000-11-17 2003-02-13 Leica Microsystems Vorrichtung zum Aufnehmen von Mirodissektaten
JP4987885B2 (ja) * 2006-03-09 2012-07-25 エージェンシー フォー サイエンス，テクノロジー アンド リサーチ 小滴中で反応を行うための装置及びその使用方法
IL288209B2 (en) 2019-05-24 2024-01-01 Berkeley Lights Inc Systems and methods for optimizing device system workflow

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US5843657A (en) * 1994-03-01 1998-12-01 The United States Of America As Represented By The Department Of Health And Human Services Isolation of cellular material under microscopic visualization

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Title
See references of WO0005587A3 *

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MXPA01000691A (es) 2002-04-08
CA2338246A1 (fr) 2000-02-03
WO2000005587A2 (fr) 2000-02-03
WO2000005587A3 (fr) 2000-04-27
AU5124499A (en) 2000-02-14
JP2002521668A (ja) 2002-07-16

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