Note: Descriptions are shown in the official language in which they were submitted.
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FLUIDIC EXTRACTION OF MICRODISSECTED SAMPLES
CROSS-REFERENCE TO RELATED APPLICATION
This application is a continuation-in-part under 35 U.S.C. ~ 120 of
copending U.S. Ser. No. 60/093,744, filed July 21, 1998; and 08/984,983, filed
December 4, 1997 the entire contents of both of which are hereby incorporated
herein by reference as if fully set forth herein.
BACKGROUND OF THE INVENTION
Field of the Invention
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.
2. Discussion of the Related Art
Prior art 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.
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This film and sample are then both dropped into the centrifuge tube where the
sample is contacted by the reaction buffer. However, this approach does not
necessarily reduce the volume of reaction buffer and/or subsequent reagents.
In addition, the previous approaches generally require sequential
handling of samples, reaction buffer and subsequent reagents in separate
apparatus, which involves many manual handling steps leading to possible
human error and relatively high cost. Therefore, what is also needed is a
solution that meets the above-discussed requirements in a more cost effective
manner.
Heretofore, the requirements of facilitating subsequent processing,
reducing the volume of reagents, and economy referred to above have not been
fully met. What is needed is a solution that simultaneously addresses all of
these requirements. The invention is directed to meeting these requirements,
among others.
SUMMARY OF THE INVENTION
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. Another embodiment of the invention is based on 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
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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 extends through said top
laminate layer and couples with said reaction chamber. 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.
These, and other, goals and embodiments of the invention will be better
appreciated and understood when considered in conjunction with the following
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description and the accompanying drawings. It should be understood, however,
that the following description, while indicating preferred embodiments of the
.
invention and numerous specific details thereof, is given by way of
illustration
and not of limitation. Many changes and modifications may be made within the
scope of the invention without departing from the spirit thereof, and the
invention includes all such modifications.
BRIEF DESCRIPTION OF THE DRAWINGS
A clear conception of the advantages and features constituting the
invention, and of the components and operation of model systems provided with
the invention, will become more readily apparent by referring to the
exemplary,
and therefore nonlimiting, embodiments illustrated in the drawings
accompanying and forming a part of this specification, wherein like reference
characters (if they occur in more than one view) designate the same parts. It
should be noted that the features illustrated in the drawings are not
necessarily
drawn to scale.
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. I 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.
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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
S 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 f lm 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. I2 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. 1 SB 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-1 SC together with a microdissected sample carrier, representing an
embodiment of the invention.
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FIG. lb 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. 21A 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. 21 C illustrates a top schematic view of the top laminate of a single
stage microdissected sample extraction device, representing an embodiment of
the invention.
FIG. 21 D illustrates a top schematic view of the spacer of a single stage
microdissected sample extraction device, representing an embodiment of the
invention.
FIG. 21 E 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 21 E, representing an embodiment of the
invention.
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FIG. 23A 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. 23C 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.
DESCRIPTION OF PREFERRED EMBODIMENTS
The invention and the various features and advantageous details thereof
are explained more fully with reference to the nonlimiting embodiments that
are
illustrated in the accompanying drawings and detailed in the following
description. Descriptions of well known components and processing techniques
are omitted so as not to unnecessarily obscure the invention in detail.
The invention includes any laminated film device mated with any
microdissected sample carrier to form a fluidic circuit. The laminated film
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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. By first aligning the
microdissected
sample that is being carried by the sample carrier within the interior of the
spacer ring, and then mating the sample carrier with the spacer ring, the
microdissected sample on the surface of the carrier can be cleanly introduced
into the extraction chamber. Before use, the extraction chamber can be kept
clean by providing a release layer on the mating surface of the 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 junctions) and/or fill ports) 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) can make the operation of such a
bubble
pump more effective, reliable and predictable. 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) can be driven through the capillaries by
capillary force(s), pumping forces) and/or acceleration forces) (e.g.,
centripetal
acceleration from a laboratory centrifuge into which the entire extraction
device
can be placed). To fill and/or empty a chamber, a pump or acceleration force
is
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usually required. 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. In
this manner fluids can be drawn in to a certain portion of the device for
digestion or incubation and then at a later time the fluids can be moved into
a
different portion of the device by applying said forces for subsequent
dilution or
analysis.
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), wells) and/or stop
junctions)
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. In addition to being mated to the sample
fzlm
carrier, the extraction device can be coupled to other analysis equipment such
as
a filter, a hybridization chamber, a PCR chamber, assay equipment, etceteras.
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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., 25W C02 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.
However, 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
1 S 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.
However, the particular material selected for laminated extraction
devices is not essential to the invention, as long as it provides the
described
function. Normally, those who make or use the invention will select the best
commercially available material based upon the economics of cost and
availability, the expected application requirements of the final product, and
the
demands of the overall manufacturing process.
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
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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. It is
desirable to have a simple, convenient method to remove the reaction buffer
from the film carrier prior to extracting the film carrier so that the liquid
is not
lifted from the reaction vessel when the film carrier is removed from the
buffer.
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.
Referring to FIGS. 1-8, the processing steps include (a) applying the
buffer to the top of the insert 100, as shown in FIGS l and 4. The buffer is
prevented from penetrating the insert 100 by the stop junction forces. Then
(b)
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 S. Extraction takes place with the apparatus in
the configuration illustrated in FIGS. 2 and 5. After extraction, 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.
Alternative Embodiments
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
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accomplished by providing a laser capture microdissection (LCM) film carrier
as part of a capillary assembly.
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
IO are applied to the mating surface prior to sealing with the film Garner.
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. Or alternatively, 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.
Turning to FIGS. 12-I4, the alternative embodiment is shown embodied
in combination with a microcentrifuge tube assembly. In FIG. 12, 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
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velocity to allow fluid to pass through stop junction and rest in the bottom
of
tube. This technique will also work with microtiter plates.
EXAMPLES
Specific embodiments of the invention will now be further described by
the following, nonlimiting examples which will serve to illustrate in some
detail
various features of significance. The examples are intended merely to
facilitate
an understanding of ways in which the invention may be practiced and to
further
enable those of skill in the art to practice the invention. Accordingly, the
examples should not be construed as limiting the scope of the invention.
Example 1
For a typical geometry it can be assumed that the film carrier has
dimensions of 1 cm. by I cm. and that the double sided tape thickness is 100
microns. The resulting enclosed volume will be only 10 mm' or 10 microliters.
At a rotational velocity of 1,000 rpm-30,000 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. I SA-15D, a single stage extraction device with a
pump is depicted. Referring to FIG. I SA, this single stage extraction device
includes a base laminate 1510. The base laminate 1510 includes an exit port
1520.
Refernng to FIG. ISB, 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 I 540 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.
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Referring to FIG. 15C, 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
S 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.
Referring to FIG. 15D, 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 operation of this single stage extraction device will now be
described. 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. After the extraction fluid has had
sufficient
time to react with 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
Refernng 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.
Referring to FIG. 21 E, 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
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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
2180. The spacer 2170 includes a mating surface 2190. The mating surface
2190 is for attachment (mating) to a biological sample earner (not shown in
FIG. 21E), for example, a laser capture microdissection transfer film carrier.
Referring to FIGS. 21 A-21 E, the component parts of the single stage
extraction device are depicted. Referring to FIG. 21A, the bottom Laminate
reflects the outline of the device and includes no specific additional
structural
features. Referring to FIG. 21 B, 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 2I 10,
the
first capillary 2130, and the second capillary 2140 can all be seen in FIG.
18.
Referring to FIG. 21 C, 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. 21 B. Similarly, the fill port hole 2155 in FIG. 21
C
aligns with the fill port 2110 in FIG. 21B.
Referring to FIG. 2ID, a spacer 2170 in the form of a ring is depicted.
The spacer 2170 includes a microdissected sample film carrier mating surface
2175.
Referring to FIG. 16-20, a series of laminate stock strips are depicted.
Referring to FIG. 16, 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. For example a suitable hydrophilic polymer are those
manufactured to have anti-fog properties.
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Referring to 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. Of course, the invention is not limited to any of the specific
dimensions. 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.
Referring to FIG. 18, a laminate stock strip for the middle laminate is
depicted. The middle laminate includes four tooling pin holes I 810 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.
Referring to 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. Again, a variety of exemplary dimensions are depicted in FIG. 19
and the invention is not limited to these dimensions. In more detail, 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.
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Referring to 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.
The operation of this single stage extraction device will now be
described. 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.
Alternatively, By placing the composite system into a microcentrifuge tube and
spinning, 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. By increasing the rpm of the centrifuge ,
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. Since the entire assembly has been previously
placed in the microcentrifuge tube, fluid carrying digested sample which
passes
out of the tip 2190 will be caught and captured in the microcentrifuge tube.
Extraction devices of this type can be termed "darts" because of their overall
appearance.
Referring to FIG. 22, a number of exemplary dimensions relating to this
single stage extraction device are shown. Of course, the invention is not
limited
to any specific dimensions. It can be appreciated that reaction buffer from
fill
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port 2110 will be contained by spacer 2170 and eventually pass through the
second capillary 2140.
Example 4
Refernng to FIGS. 23A-23G, a two stage extraction device is depicted.
Referring to FIG. 23A, in this embodiment, 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.
Referring to FIG. 23B, 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.
Referring to FIG. 23C, 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.
Referring to FIG. 23D, 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.
Refernng to FIG. 23E, a layer of foam 2380 with pressure sensitive
adhesive on both sides has a hole 2382. The hole 2380 will define a dilution
chamber. Referring to FIG. 23F, a cover layer 2390 is depicted. The cover
layer 2390 is placed on top of foam 2380.
Referring now to FIG. 23G, the assembled two stage extraction device is
depicted. FIG. 23G shows a number of exemplary dimensions associated with
the two stage extraction device. Of course, the invention is not limited to
any
particular dimensions.
The operations) of this two stage extraction device will now be
described. 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. When ready for use, the release layer 2372 is removed from the
spacer 2370 and a sample film carrier (not shown) is mated with the
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microdissected sample film Garner 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. After
extraction is complete, 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. In this
way,
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. By continuing to actuate the second pump, 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.
Practical Applications of the Invention
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.
Advantages of the Invention
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
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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.
All the disclosed embodiments of the invention described herein can be
realized and practiced without undue experimentation. Although the best mode
of carrying out the invention contemplated by the inventors is disclosed
above,
practice of the invention is not limited thereto. Accordingly, it will be
appreciated by those skilled in the art that the invention may be practiced
otherwise than as specifically described herein.
For example, the individual components need not be formed in the
disclosed shapes, or assembled in the disclosed configuration, but could be
provided in virtually any shape, and assembled in virtually any configuration.
Further, the individual components need not be fabricated from the disclosed
materials, but could be fabricated from virtually any suitable materials.
Further,
although the extraction devices described herein can be physically separate
modules, it will be manifest that the extraction devices may be integrated
into
the apparatus with which they are associated. Furthermore, all the disclosed
elements and features of each disclosed embodiment can be combined with, or
substituted for, the disclosed elements and features of every other disclosed
embodiment except where such elements or features are mutually exclusive.
It will be manifest that various additions, modifications and
rearrangements of the features of the invention may be made without deviating
from the spirit and scope of the underlying inventive concept. It is intended
that
the scope of the invention as defined by the appended claims and their
equivalents cover all such additions, modifications, and rearrangements. The
appended claims are not to be interpreted as including means-plus-function
limitations, unless such a limitation is explicitly recited in a given claim
using
the phrase "means-for." Expedient embodiments of the invention are
differentiated by the appended subclaims.
