U.S. patent application number 13/439506 was filed with the patent office on 2012-08-09 for fluid exchange in a chamber on a microscope slide.
This patent application is currently assigned to Dako Denmark A/S. Invention is credited to Steven A. Bogen, Herbert H. Loeffler.
Application Number | 20120201723 13/439506 |
Document ID | / |
Family ID | 29738802 |
Filed Date | 2012-08-09 |
United States Patent
Application |
20120201723 |
Kind Code |
A1 |
Loeffler; Herbert H. ; et
al. |
August 9, 2012 |
FLUID EXCHANGE IN A CHAMBER ON A MICROSCOPE SLIDE
Abstract
A sample chamber is formed by a housing sealed against a
microscope slide. The housing has fluid ports, including a well
formed over at least one port. In a rinse station, rinse solution
is drawn from a reservoir through the chamber to a waste reservoir.
At a fill station, an aliquot of reagent already placed in the well
is driven into the chamber. The reagent may be driven into the
chamber by first drawing a vacuum on the chamber through the
aliquot of reagent and then releasing the reagent to be drawn into
the chamber by the vacuum.
Inventors: |
Loeffler; Herbert H.;
(Arlington, MA) ; Bogen; Steven A.; (Sharon,
MA) |
Assignee: |
Dako Denmark A/S
|
Family ID: |
29738802 |
Appl. No.: |
13/439506 |
Filed: |
April 4, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11895872 |
Aug 28, 2007 |
8173068 |
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13439506 |
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10696219 |
Oct 29, 2003 |
7318913 |
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11895872 |
|
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|
09549414 |
Apr 14, 2000 |
6673620 |
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10696219 |
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60130171 |
Apr 20, 1999 |
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Current U.S.
Class: |
422/501 |
Current CPC
Class: |
G01N 2035/1032 20130101;
G02B 21/26 20130101; B01L 2200/0642 20130101; B01L 2300/0822
20130101; G02B 21/34 20130101; Y10T 436/25 20150115; G01N 1/312
20130101; G01N 2035/0437 20130101; B01L 2200/027 20130101; G01N
35/00029 20130101; B01L 3/502 20130101; Y10T 436/112499 20150115;
G01N 2035/00039 20130101; B01L 2400/049 20130101; B01L 2300/0877
20130101; Y10T 436/2575 20150115 |
Class at
Publication: |
422/501 |
International
Class: |
B01L 3/00 20060101
B01L003/00 |
Goverment Interests
GOVERNMENT SUPPORT
[0003] The invention was supported, in whole or in part, by NIH
grant 1R43CA84686-01 from Public Health Service. The Government has
certain rights in the invention.
Claims
1. An apparatus for adding and removing liquid reagent to and from
a sample comprising: a microscope slide supporting the sample; a
chamber forming a cavity over said microscope slide, said chamber
being releasably sealed to said microscope slide; a fluid port in
the wall of said chamber through which fluids can be added or
removed, said fluid port having a valve seat, said fluid port
further comprising a reagent well capable of retaining an entire
aliquot of reagent prior to the reagent passing into the cavity; a
normally closed valve comprising a moveable valve element that, in
a closed position, presses against the valve seat of the fluid
port, said valve capable of opening by movement of the moveable
valve element away from the valve seat to admit fluid through the
port between the valve seat and the moveable valve element into the
chamber; a flexible membrane in said valve, the flexible membrane
being an extension of a gasket that surrounds the chamber and seals
against the microscope slide; a conduit through which a source of
negative or positive air pressure can be communicated; a member
associated with the conduit capable of moving the moveable valve
element away from the valve seat to open said valve to admit fluid
through the port into the chamber; and an actuator capable of
causing relative movement between the fluid port and conduit to
cause the member to move the moveable valve element away from the
valve seat and to engage said conduit and fluid port to each other
so that the two are in fluid communication with each other.
2-10. (canceled)
Description
RELATED APPLICATION(S)
[0001] This application is a continuation of U.S. application Ser.
No. 10/696,219, filed Oct. 29, 2003, which is a Divisional of U.S.
application Ser. No. 09/549,414, filed Apr. 14, 2000, which claims
the benefit of U.S. Provisional Application No. 60/130,171, filed
Apr. 20, 1999.
[0002] The entire teachings of the above application(s) are
incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0004] This invention relates to an instrument that is capable of
performing incubations of small volumes of reagents on the surface
of a microscope slide. A variety of assays are typically carried
out on the surface of a microscope slide. These assays generally
aim to determine if a suspected analyte is present in a patient
biopsy. They include: (1) in situ hybridization, for the detection
of nucleic acid targets in a tissue or cell sample, (2)
immunohistochemistry, for the detection of specific proteins in a
tissue or cell sample, (3) histochemical stains, for the detection
of certain types of chemical compounds or classes of microorganisms
in a tissue sample. In addition, there are two other types of
assays that are often carried out on the surface of a glass slide.
Rather than testing for the presence of an analyte in a tissue
biopsy, these assays aim to detect specific molecules in a
solution. They are (1) gene arrays, whereby an array of known
nucleic acid targets are immobilized directly on the glass slide,
and (2) protein arrays, whereby an array of known proteins are
immobilized on the glass slide.
[0005] In each of these instances, a glass slide serves as the
preferred support on which the assay is carried out. The reason a
glass slide is used is that it is optically clear and flat. These
physical properties facilitate the ability of an instrument, such
as a microscope, to optically detect a fluorescent or colorimetric
signal. In order to highlight certain desired features, the above
assays require that the.slides be treated with a series of reagent
incubations. Each incubation needs to occur for a specific time
(typically 15-60 minutes) and at a specified temperature
(typically, from room temperature to 95.degree. C.). The optical
advantages of a microscope slide are somewhat counterbalanced by
certain difficulties in performing the assay. The treatment of the
tissue sections on a microscope slide for the purpose of
highlighting certain histologic features is often called
"staining." Since the surface of the slide is flat, reagent can
easily drain off the edge of the microscope slide, especially if
the slide is not perfectly level. Moreover, the large surface area
to volume ratio of reagent spread over the slide surface promotes
evaporation. Evaporation of reagent interferes with the performance
of the assay. If the reagent evaporates, then it will not
continuously contact the tissue sample. Drying artifacts may cause
the assay result ("stain") to not be accurate. Lastly, it is
important to spread the reagent over the slide surface. Surfactants
are sometimes used to promote reagent spreading. If the reagent
does not spread, then the reaction may fail to occur over all of
the tissue biopsy, or over all portions of the array. Therefore,
the prior art comprises a great number of attempts to construct
apparatus and devices that aim to facilitate or automate the sample
preparation/treatment steps of a biological sample on a glass
slide.
[0006] The general approach to solving these problems in the past
has been to enclose an area of the slide surface, forming a
chamber. Desirable features for such a chamber are: [0007] a)
Liquid spreading. Reagents must be evenly spread, without entrapped
air bubbles. [0008] b) Use of minimal reagent volume (ideally less
than 100 microliters to cover the slide surface). [0009] c) Prevent
evaporation when the reagent is heated to 95.degree. C. [0010] d)
Automatic reagent injection and removal. Namely, the apparatus
needs to be compatible with an automated fluid transfer system.
[0011] e) Protection of the tissue section against physical
damage.
[0012] One method of addressing at least some of the requirements
described above is to entrap reagent under a coverslip. For in situ
hybridization procedures, reagent is conventionally placed directly
on top of the tissue section with a pipette and covered with a
coverslip. The edges of the coverslip are then sealed with nail
polish or rubber glue. The coverslip both spreads out the reagent
into a relatively uniform layer and prevents evaporation. It is
important to avoid entrapping air bubbles under the coverslip.
Otherwise, there will be an area of the tissue section that does
not contact the hybridization solution.
[0013] No existing technology is suited to automating coverslipping
for in situ hybridization. Coverslips can be applied to slides in
an automated fashion; several companies serving the histopathology
market sell dedicated coverslipping machines. However, such
coverslipping machines are not likely to be adaptable to this
application, because (i) it will be difficult to automate sealing
the coverslip edges, such as with glue, and (ii) it will be
difficult to robotically remove the coverslips without damaging the
tissue section.
[0014] An alternative method for spreading small amounts of
reagents was described in 1988, by Unger, et.al. (Unger, E R, D J
Brigati, M L Chenggis, et. al. 1988. Automation of in situ
hybridization: Application of the capillary action robotic
workstation. J. Histotechnology 11:253-258.) Specifically, they
described a modification of the Code-On slide stainer for use with
in situ hybridization. Instead of a coverslip, two slides were
placed in close apposition to each other, forming a capillary gap.
Liquids, such as a hybridization solution, could then be applied to
the bottom of the gap. The reagents "wicked" in by capillary
action. Placing the slides in a heated humidity chamber prevented
evaporation. The Code-On worked well in the right hands, but was
labor-intensive and required a great deal of experience and care
each day in setting it up. Slight scratches or imperfections in the
surface of the glass slide sometimes caused air bubbles to be
entrapped in the capillary gap, resulting in areas of the tissue
not being stained.
[0015] An early description of a slide chamber is disclosed in U.S.
Pat. Nos. 4,847,208 and 5,073,504. A chamber was apposed to the
surface of a slide, forming walls capable of preventing lateral
spillover of reagent. Moreover, a hinged cover minimized the
evaporative loss of reagent. Each chamber included a fluid inlet
and outlet port.
[0016] WO99/34190 discloses a chamber formed by the insertion of a
microscope slide into a cartridge device. Reagent is dispensed onto
a portion of the slide that protrudes from the cartridge and is
caused to flow into a capillary gap-type space by moving the slide
inwards. The chamber is not sealed from the outside environment.
Therefore, reagent will be expected to evaporate, especially if the
samples are heated. Moreover, reagent can flow around and
underneath the slide, thereby increasing the volume requirement to
cover the slide surface. Lastly, it is unclear how often bubbles
will be entrapped over the slide surface, since no specific
mechanism in the cartridge prevents them.
[0017] A review of other methods of forming a chamber, and their
drawbacks, are also described in the Background section of
WO99/34190.
SUMMARY OF THE INVENTION
[0018] In embodiments of the present invention, a fluid handling
apparatus is capable of spreading small amounts of liquid reagent
over a flat surface, such as a microscope glass slide. The reagent
may be sealed within a confined cavity, or "chamber", so as to
prevent evaporation even with heating of small amounts of reagent
during an incubation period. One surface of this chamber is the
flat slide surface. The remaining surfaces are formed by a cell.
The cell is preferably a plastic disposable part that fits on top
of the slide, over the area containing the tissue, biologic cells,
or array mounted on the glass slide. The cell forms a fluid seal to
the surface of the glass by means of a gasket. The gasket is
mounted in a recess on the face of the cell that mates with the
glass slide.
[0019] Each cell has two fluid ports. These ports are in fluid
communication with the chamber. Therefore, when liquid reagent is
inserted into a fluid port, it can travel into the chamber and
contact the biologic sample or array mounted on the glass surface.
The fluid ports on a cell are preferably positioned on opposite
ends of the cell. This allows for liquid reagent or wash solution
to flow in one fluid port, fill the chamber, and then exit the
other fluid port at the other end of the cell. Each fluid port has
a valve occluding the orifice that is normally closed.
Consequently, the chamber is sealed. Unless the valves are opened,
the chamber is normally not accessible to the outside environment.
Liquid reagents or wash solutions are only added or removed by
opening one or both of the valves associated with the fluid ports.
The fact that the fluid ports are normally sealed by valves helps
prevent evaporation.
[0020] The instrument comprises a plurality of positions for glass
slides. Each position has a mechanism to clamp a cell to the glass
slide. Keeping the cell and slide apposed to each other in a fixed
spatial relationship is important in forming a sealed chamber for
reagent incubations. Reagents and wash solutions are added and
removed by the use of two liquid handling "stations". In the
illustrated embodiment, the liquid handling stations move over
non-moving slides. It is also conceivable, and ultimately preferred
in an automated instrument, to reverse that relationship to
automate the staining process by moving the slides, such as on a
rotary carousel. The slides would move past non-moving liquid
handling stations positioned on the periphery of the rotary
carousel of slides. This type of arrangement is described in U.S.
Pat. No. 5,947,167 by the same inventors which is incorporated by
reference in its entirety.
[0021] The method of adding and subsequently washing reagents out
from the chamber is an important part of this system. Washing
reagent out from the chamber is required after an incubation, to
thoroughly remove any unreacted reagent before the subsequent
treatment step. Washing usually involves treating the biologic
sample mounted on the glass slide with an excess of a buffer or
wash solution. The unreacted reagent is diluted in the excess
volume of the wash solution and removed by aspirating away the wash
solution. In this system, washing the biologic sample involves
flushing the chamber containing the biologic sample with the wash
solution. Wash solution is pumped in one fluid port and removed
from the other. This process requires the presence of a "fluid
injector" and a "fluid aspirator", each articulating with a fluid
port. Each injector or aspirator has a piston that is capable of
opening the valve positioned in the fluid port. The piston opens
the valve by deflecting an elastomeric seal that occludes the
orifice of the fluid port. For the purpose of washing the biologic
sample, a fluid injector pushes wash solution into the chamber. The
fluid aspirator captures the reagent after it has passed through
the chamber. The fluid aspirator can then channel the waste wash
fluid to one or more reservoirs for ultimate disposal. This washing
process occurs at a "wash station" that has one fluid injector and
one fluid aspirator. The injector and aspirator are mounted on to a
mechanism that lowers and raises them together.
[0022] Reagent injection into the chamber occurs at a separate
"reagent injection station". Two different methods for injecting
reagent will be described. The first step for both methods is that
a small aliquot of the desired reagent to be injected is placed in
a reagent well at the fluid inlet port. In the first method, the
reagent injection station includes only a fluid injector. The fluid
injector is mounted on to a mechanism that lowers the injector so
that the injector articulates with the fluid port on the cell. As
the fluid injector is lowered, it first forms a fluid-tight seal to
the fluid port. The piston on the injector then opens the valve in
the fluid port. A strong momentary vacuum is then drawn through the
fluid injector. The vacuum is transmitted through the fluid port
into the chamber. The small amount of air in the chamber bubbles up
through the reagent in the reagent well. The vacuum in the fluid
injector is then released, returning the pressure above the reagent
to atmospheric level. The reagent is then drawn into the chamber by
the strong residual vacuum in the chamber. Bubbles do not form
because there is little to no air in the chamber to form an air
bubble. Alternatively, the vacuum can be drawn through a second
port which is closed before the port to the aliquot is opened. In
either approach, the liquid is drawn into but not through the
chamber by the vacuum.
[0023] In another method of fluid injection, reagent is placed into
the reagent well, as before. A fluid injector is positioned above
the fluid inlet port. In addition, the fluid aspirator is
positioned above the fluid outlet port. The valves of both fluid
ports are opened by this process. Reagent is then pushed into the
chamber by a burst of air pressure. The transient, high-pressure
reagent injection avoids entrapping bubbles by forcing laminar flow
of reagent through the chamber. Once the reagent completely fills
the chamber, the pressure is removed and the valves are closed by
disengaging the fluid injector and fluid aspirator.
[0024] Thus, in accordance with one aspect of the invention, an
apparatus for adding and removing liquid reagents to and from a
sample comprises a flat surface supporting the sample and a chamber
forming a cavity on the flat surface, the chamber being releasably
sealed to the flat surface. Fluids can be added or removed through
a fluid port in the wall of the chamber. A source of negative or
positive air pressure is provided in a conduit, and an actuator is
able to move the fluid port and conduit relative to each other to
engage the conduit and fluid ports to each other so that the two
are in fluid communication.
[0025] The chamber may include a valve that is positioned at the
fluid port, and the conduit may further include a piston capable of
opening the valve when the conduit and port are in communication
with each other. A preferred valve is a flexible element below the
port which is an extension of a gasket of the chamber which seals
against the flat surface.
[0026] A well capable of holding an aliquot of reagent may be
provided over the fluid port. Multiple chambers may be moved
relative to the actuator to position a selected chamber at the
actuator.
[0027] Another aspect of the invention includes novel methods of
applying reagent to a sample. In one method, a vacuum is applied to
the chamber in which the sample is positioned. After application of
the vacuum ceases, the reagent is allowed to be drawn into the
chamber by the vacuum formed within the chamber. In a preferred
approach, the vacuum is applied to the chamber through an aliquot
of reagent held in a well. When application of the vacuum above the
aliquot ceases, the aliquot is drawn into the chamber.
[0028] In accordance with another novel method, a sealed chamber is
apposed to a flat surface supporting the sample. An aliquot of
reagent is dispensed into a reagent well located above a fluid port
in the chamber. A source of air pressure is applied to the fluid
port to cause the reagent to be pushed through the fluid port into
the chamber.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] The foregoing and other objects, features and advantages of
the invention will be apparent from the following more particular
description of preferred embodiments of the invention, as
illustrated in the accompanying drawings in which like reference
characters refer to the same parts throughout the different views.
The drawings are not necessarily to scale, emphasis instead being
placed upon illustrating the principles of the invention.
[0030] FIG. 1 is an exploded perspective drawing of the components
comprising a slide nest.
[0031] FIG. 2 is a perspective drawing of an assembled slide
nest.
[0032] FIG. 3 is an exploded end view of the components of a slide
nest.
[0033] FIG. 4 is an exploded cross-sectional view of the slide nest
cut through the lines A-A, as indicated in FIG. 3.
[0034] FIG. 5 is a cross-sectional view of the slide nest, fully
assembled.
[0035] FIG. 6 is a high magnification cross-sectional view of the
circled area in FIG. 5.
[0036] FIG. 7 is a high magnification cross-sectional view of the
seal and its physical relationship to the underlying slide and
overlying plastic cover.
[0037] FIG. 8 is a schematic representation of an alternative,
non-preferred embodiment of a seal. It is illustrated to highlight
the advantages of the preferred embodiment of the seal shown in
FIG. 7.
[0038] FIG. 9 is a cross-sectional representation of a fluid
injector.
[0039] FIG. 10 is a cross-sectional representation of a fluid
injector and fluid port of an ISH cell that are apposed to each
other.
[0040] FIG. 11 is a perspective view of a slide staining
instrument.
[0041] FIG. 12 is a schematic representation of the fluid pathways
found in the slide staining instrument.
[0042] FIG. 13 is a schematic representation of the fluid pathway
for reagent filling, in an alternative embodiment.
DETAILED DESCRIPTION OF THE INVENTION
[0043] A description of preferred embodiments of the invention
follows.
[0044] FIG. 1 shows an exploded view of a complete slide nest
assembly. The assembly was designed as an "ISH cell", "ISH"
standing for in situ hybridization; however, it can be readily
applied to other applications such as outlined in the Background.
The ISH cell is comprised of a plastic cover 1 and a molded gasket
3. The gasket 3 fits into a groove (not shown) on the underside of
the plastic cover 1. The ISH cell is positioned on top of a
microscope slide 9 that bears a biologic sample 5 mounted on the
surface of the slide. The microscope slide 9 rests on a heater
plate 7. The heater plate is mounted, with screws (not shown), into
a slide nest base 11. A resistive heating element (not shown) is
attached to the underside of the heater plate 7. The heater plate
thereby protects the electrical heater from any liquids that might
spill. More importantly, the heater plate 7 diffuses the heat that
emanates from the heating element to form an evenly heated surface.
The thermal mass of the heater plate 7 also serves to stabilize the
temperature around a desired mean temperature. Without sufficient
thermal mass, actuation of the heating element can cause the
temperature to overshoot the desired temperature. With some added
thermal mass, as associated with the heater plate 7, the
temperature rises more slowly than it would otherwise after the
heating element is actuated.
[0045] Also shown in FIG. 1 is a clamping mechanism for keeping the
plastic cover 1, gasket 3, and microscope slide 9 tightly apposed
to each other. This clamping mechanism is important in maintaining
a fluid- and air-tight seal between the plastic cover 1, gasket 3,
and microscope slide 9. The clamping mechanism is formed by the
hinged cover 17, foam spring 13, and latch 15. When the elements
shown in FIG. 1 are fully assembled, the hinged metal clamp 17 is
closed so that the end of the clamp 17 is captured by the latch 15.
The clamp 17 compresses the foam spring 13 which, in turn, presses
downwards on the plastic cover 1, gasket 3, and slide 9. The foam
spring 13 is inserted to allow for minor variability in the
dimensions of the parts shown in FIG. 1. Without the foam spring
13, too little pressure might be applied, thereby failing to form a
seal. Alternatively, too much pressure might be applied, causing
the microscope glass slide 9 to crack. By using the foam spring 13,
the clamping mechanism is designed to slightly overcompress. The
compressibility of the foam spring 13 serves to buffer that
compressive force applied by the clamping mechanism, preventing the
slide 9 from cracking.
[0046] FIG. 2 shows the slide nest fully assembled. The clamping
mechanism formed by the hinged metal clamp 17, the foam spring 13,
and the latch 15 is actively maintaining the parts in fixed
apposition to each other. Two fluid ports 19 and 21 protrude above
the hinged metal clamp 17. An aperture 2 in the plastic cover 1
allows a direct view of a portion of the underlying microscope
slide 9. The ability to view the slide 9 is for the purpose of
viewing patient or sample information that might be placed on one
end of the slide 9. In addition, it allows a bar code reader (not
shown) to be able to view a bar code (not shown) that might be
placed on one end of the glass slide 9.
[0047] FIGS. 3 and 4 are end and cross-sectional exploded views of
the components shown in FIG. 1. These views demonstrate the
presence of two elastomeric valve stems 4a and 4b that are part of
the same molded gasket 3. The valve stems 4a and 4b fit into valve
seats 6a and 6b, respectively. Valve seats 6a and 6b are formed as
recesses in the underside of the plastic cover 1. When the valve
stems 4a and 4b are inserted into valve seats 6a and 6b, the valve
stems occlude fluid or air flow into or out of the fluid ports 19
and 21.
[0048] A better understanding of the operation of the valves
associated with each of the fluid ports 19 and 21 can be obtained
by reference to FIGS. 5 and 6. FIG. 5 is a cross-sectional view of
the slide nest where the plane of section cuts through the valve
stems 4a and 4b. A high magnification cross-section of the circled
area of FIG. 5 is shown in FIG. 6. When fully assembled, the valve
stem 4a fits into valve seat 6a and occludes the lowermost aspect
of the neck 14 of fluid port 19. The neck 14 sits at the base of a
funnel-shaped fluid port 19. When reagent is added to the fluid
port 19, the funnel shape causes the reagent to collect at the
bottom of the fluid port 19, towards the neck 14. Reagent can not
travel past the neck 14 because the valve stem 4a blocks further
flow. If the valves stem 4a were to be deflected, then reagent
would be able to enter communicating passageway 23. That passageway
23 leads to the underside of the plastic cover 1 and the microscope
slide 9.
[0049] An even higher magnification cross-sectional view of the
communicating passageway 23, the gasket 3, and the chamber is shown
in FIG. 7. The chamber is laterally enclosed by a perimeter formed
by the gasket 3. In the illustrated embodiment, the chamber is
oval-shaped. However, it can be of any convenient shape. The roof
of the chamber is formed by a chamber upper surface 10 that is, in
fact, the undersurface of the plastic cover 1. The chamber upper
surface 10 is slightly recessed (approximately 3/1000 of an inch)
relative to the undersurface of the plastic cover 1 that is not
encircled by the gasket 3. The lower chamber surface 12 is, in
fact, the surface of the glass slide 9. Tissue sections are
typically much thinner than the height of the chamber which is
preferably in the range of about 3/1000 to 6/1000 inch.
[0050] The gasket 3 fits into a gasket recess 16 that is on the
underside of the plastic cover 1. The gasket 3 has a lip 18 that
forms an interference fit between the upper chamber surface 10 and
the lower chamber surface 12. This feature is important in limiting
the volume of reagent by preventing the reagent from ever reaching
the gasket recess 16 which serves to anchor the gasket 3. An
acceptable, though non-preferred alternative sealing method is
illustrated in FIG. 8 to illustrate the advantage of that shown in
FIG. 7. If an O-ring 20 were used as a seal, then reagent would
gain entry to the gasket recess 16 as illustrated by the bold arrow
in FIG. 8. Some reagent would be wasted in filling the void volume
of the gasket recess 16. Most of the volume of the gasket recess 16
would be occupied by the O-ring 20 itself. However, the gasket
recess 16 is necessarily larger than the O ring 20 because the
O-ring 20 needs to deform as pressure is applied. Our testing
disclosed that the design of FIG. 7 is superior in limiting the
amount of reagent that is required to fill the chamber.
[0051] FIG. 9 is a cross-sectional representation of a fluid
injector 25. In the illustrated and preferred embodiment, the same
design is used for a fluid aspirator. The fluid injector 25 is
comprised of an injector housing 27 into which a metal threaded
shaft 29 is inserted. The shaft 29 fits into a hollow central core
of the injector housing 27. Clockwise rotation of the shaft 29
causes the shaft 29 to move downwards, deeper into the hollow
central core of the injector housing 27. Counterclockwise rotation
causes the shaft 29 to move upwards. A groove 31 is provided at the
top of the shaft 29 so that it can be rotated with a screwdriver.
The threaded interface between the shaft 29 and the injector
housing 27 is fluid- and air-tight. Consequently, air or liquid
above the shaft 29 can not communicate with air or liquid around
the lower portion of the shaft 29, below the threaded interface.
The shaft narrows to form a piston 33 that protrudes from a lower
face 35 of the injector housing 27. There is a small gap between
the piston 33 and the orifice in the lower face 35 through which
the piston 33 protrudes.
[0052] The fluid injector 25 also includes an air or fluid pathway,
comprising a hollow side port 39 that communicates with an injector
cavity 37. The cavity 37, in turn, is in communication with the
orifice in the lower face 35 through which the piston 33 protrudes.
The fluid injector 25 is constructed so that air or fluid pressure
applied to the side port 39 will be transmitted to the orifice in
the lower face 35 through which the piston 33 protrudes. Such air
or fluid pressure does not travel beyond the threaded interface
between the shaft 29 and the plastic body 27 because the interface
is air- and fluid-tight.
[0053] A elastomeric O-ring 41 is mounted towards the bottom of the
fluid injector 25. This O-ring 41 is capable of forming an air- and
fluid-tight seal when compressed against a conforming surface such
as a fluid port 19 or 21. FIG. 10 shows the relationship of the
fluid injector 25 to the fluid port 19 when the two are apposed to
each other. The O-ring seal 41 compresses against the fluid port,
forming an air- and fluid-tight seal. The piston 33 compresses the
valve stem 4a, thereby deflecting it away from the fluid port neck
14. This opens the valve and places the hollow side port 39 in
fluid and air communication with the communicating passageway 23
and the chamber.
[0054] FIG. 11 is a perspective representation of an instrument 43
that incorporates positions for eight slides. The instrument 43 is
shown with ISH cells in each of the eight positions. Each of the
hinged covers 17 is clamped downwards underneath the latch 15. A
heater controller pad 45 is located on the front panel of the
instrument 43.
[0055] The heater controller pad allows someone using the
instrument 43 to enter a desired temperature to which the heaters
will be heated. Switches 47 are also provided to turn off the heat
to any slide positions that are empty. It is also envisioned that
heater control circuitry can be incorporated that will allow each
heater to be heated to a temperature distinct from that of other
heaters. Such circuitry is described in U.S. Pat. No. 5,645,114 and
U.S. patent application Ser. No. 09/032,676, filed Feb. 27, 1998,
both of which are incorporated herein by reference in their
entireties. The instrument 43 also comprises a moving platform 49
that slides from side to side on a track 51. The moving platform
has two actuators 53 and 55. Actuator 53 is termed the "rinse
actuator", and is connected to two fluid injectors by means of a
mechanical linkage 57, and the linkage 57 is normally kept in an up
position by two springs 59 that are mounted underneath the linkage
57.
[0056] Actuator 55 is termed the "fill" actuator and may be
connected to one or two fluid injectors, depending upon the method
of filling the chamber with reagent (to be described later). In
each method, an aliquot of reagent is preferably placed in the well
of port 19, as by an automatic pipette, before the assembly is
positioned below the fill activator. If only one fluid injector is
used, then a dummy injector is used in lieu of the absent fluid
injector. The dummy injector is cylindrical injector housing 27,
without the shaft 29 and piston 33. It is used in lieu of the fluid
injector to balance the distribution of downward forces generated
by the actuator 55. The actuators 53 and 55 are represented as
manually-controlled handles. However, it is understood that they
could also be operated by motors, under computer control. Actuator
55 is shown in the "down" position, causing a fluid injector and
dummy injector to be apposed to the fluid ports 19 and 21. Thus,
when the actuator 55 is in the down position, the fluid injector 25
and fluid port 19 are in the relationship as shown in FIG. 10.
[0057] Also shown in FIG. 11 are two bottles 61 and 63. These
bottles are connected, by flexible tubing (not shown), to the
valves 65, 67, and 69 which in turn are connected to fluid
injectors 25a-25c (FIG. 12) mounted on the moving platform 49. The
pathways for the fluid connections are shown in FIG. 12. The upper
half of FIG. 12 describes the fluid connections for the fill
station. The lower half of FIG. 12 describes the fluid connections
for the rinse station. Both stations require a source of vacuum
pressure, not shown in the diagrams. This source is most
conventionally a vacuum pump. A distribution manifold 71 for the
vacuum channels the vacuum force to valve 65 and to a pressure
regulator 73. Valve 65 is normally in the vent position, as shown
in FIG. 12. In both the fill and rinse stations, the fluid
injectors are represented in the down position, as illustrated in
FIG. 10. As shown in FIG. 12, the vacuum force can be transmitted
through the manifold 71 and valve 65 to the side port 39 of the
fluid injector 25a in the fill station. Alternatively, the valve 65
can vent the side port 39 of the fluid injector 25a to atmosphere.
The object represented as 25d can either be a "dummy" injector, as
previously described, or a normally constructed injector.
[0058] Vacuum force can also be transmitted through regulator 73 to
valve 67. Valve 67 provides a conduit to one side of a waste trap
bottle 61. The other side of the bottle 61 is connected, via
flexible tubing, to the side port of fluid injector 25b. Unless
manually actuated, valve 67 connects the side port 39 of fluid
injector 25b to atmosphere ("vent"). Fluid injector 25c is also
part of the rinse station. Its side port 39 is connected to valve
69 via flexible tubing. The valve can either vent the line to
atmosphere or connect it to bottle 63 filled with rinse solution.
Unless manually actuated, valve 69 is normally connected to the
bottle 63.
[0059] The method of filling and rinsing the chambers using this
apparatus will now be described. This method description assumes
that a slide 9 is inserted into a slide nest and clamped securely,
as already described. A biologic specimen 5 or array, also as
already described, is located on the surface of the slide 9. The
goal is to incubate the specimen with a reagent for a defined
period of time, as already described, and then remove that reagent
by a rinse process. This rinse process, as already described,
involves flushing the reagent away with an excess of a rinse
solution. The biologic specimen 5 is contained within a sealed
chamber, whose boundaries have already been described. In this
section, we will describe how rinsing and reagent filling is
accomplished in this context.
[0060] The explanation can best be understood with reference to
FIGS. 11-12. Rinsing a specimen 5 on a slide 9 is accomplished by
moving the moving platform 49 so that the rinse actuator 53 is
positioned over the desired slide 9. The actuator 53 is manually
depressed, causing fluid injectors 25b and 25c to be apposed to the
fluid ports 19 and 21. Manually depressing the actuator thereby
causes the pistons 33 of fluid injectors 25b and 25c to open the
valves associated with fluid ports 19 and 21. Valve 67 is then
actuated so as to connect the vacuum to the waste bottle 61. Valve
69 normally connects the rinse solution bottle 63 to the fluid
injector 25c. Actuation of valve 69 is therefore not initially
necessary. By pulling vacuum on fluid injector 25b, rinse solution
is drawn through valve 69 and fluid injector 25c, into the chamber
formed on top of the slide 9. Fluid flows in the direction of the
arrow shown at the bottom half of FIG. 12. After a sufficient
amount of rinse solution has passed through the chamber, valve 69
is actuated. This actuation will cause the vacuum force to pull
air, rather than rinse solution, through the chamber. Thus, any
rinse solution in the chamber will be aspirated, leaving an empty,
air-filled, chamber. Rinse solution is collected in the waste
bottle 61. The regulator 73 is important to limit the flow rate of
rinse solution through the chamber. If the vacuum pressure is too
high, the high flow rate of rinse solution through the chamber
might potentially shear the biologic sample 5 off of the slide
9.
[0061] There are at least two methods for filling reagent into the
chamber so as to incubate the biologic specimen 5 with the reagent.
According to the first method, fluid injector 25d is, in fact, a
"dummy" injector and does not open the valve of the underlying
fluid port 21. In this first method, an aliquot of the reagent is
manually dispensed into the fluid port 19. The conical shape of the
fluid port serves as a reservoir to retain the reagent. Because the
valve associated with the fluid port 19 is normally closed, the
reagent does not initially enter into the chamber via the
communicating passageway 23. The user then positions the moving
platform 49 so that the actuator 55 is positioned over the fluid
port 19 containing reagent. The user then depresses the actuator
55, causing fluid injector 25a, and dummy injector 25d, to mate
with fluid ports 19 and 21. When the actuator 55 is depressed,
fluid injector 25a has a piston 33 that opens the valve associated
with the fluid port 19. At this point, the user actuates valve 65,
causing a high vacuum force to be transmitted through the valve 65
and fluid injector 25a into the chamber. In order to draw a strong
vacuum inside the chamber, it is important that the gasket 3 forms
a good seal between the plastic cover 1 and the slide 9. Moreover,
it is important that the O-ring seal 41 forms a good seal between
the fluid injector 25a and the fluid port 19.
[0062] Any air inside the chamber is evacuated. The air bubbles up
through the reagent filling the fluid port 19. Although a high
vacuum is drawn, the air flow is negligible because the volume of
the chamber is only approximately 100. To minimize the flow, the
volume should preferably be less than 200 microliters. It is
necessary to draw a vacuum for, at most, 1-3 seconds.
[0063] Valve 65 is then released to its normal position, venting to
atmosphere. The reagent inside the fluid port 19 then experiences
atmospheric pressure above it and a strong negative vacuum force
below it. This pressure differential instantaneously draws the
reagent into the chamber. Since there is a near vacuum inside the
chamber, no air bubbles form inside the chamber. The reagent is
forced to evenly spread, filling the volume of the chamber. Because
the chamber self fills to eliminate the vacuum throughout the
chamber, a precise aliquot of reagent need not be supplied to the
well.
[0064] A second method for filling the chamber with reagent is
characterized in FIG. 13. It requires that fluid injector 25d has a
piston 33 capable of opening the valve of the underlying fluid port
21. According to this second method, reagent is manually added to
fluid port 19, as before. The moving platform is also moved so that
the actuator 55 is located over the fluid port 19 containing
reagent. The actuator 55 is depressed, causing valves associated
with both fluid ports 19 and 21 to open. In this second method, a
burst of pressure drives the reagent into the chamber as port 21 is
vented. Such a pressure head causes the reagent to enter the
chamber in a laminar fashion. Alternatively, a short pulse of
vacuum force could be applied to port 21 to pull reagent into the
chamber. In either case, the pressure or vacuum should be
discontinued before the reagent flows through the chamber. As yet
another alternative, the vacuum could be drawn through port 21
while the valve to port 19 is held closed. Then the valve to port
21 would be closed before opening the valve to port 19 to draw
reagent into the chamber.
[0065] In prior approaches, when the only pressure driving reagent
into a capillary-sized chamber is the wicking action of the chamber
surfaces (capillary action), then minor imperfections in the
surfaces of the chamber, or the fluid drag created by the biologic
specimen 5, can cause bubbles to form. The drag of the tissue
sample against the weak capillary flow may be sufficient to cause
the flow to pass around the sample and converge downstream of the
area of fluid drag, entrapping an air bubble over the sample. If
the pressure head driving the fluid flow is high relative to the
fluid drag, then the fluid drag represents minor resistance as
compared to the pressure head. Raising the pressure head driving
reagent into the chamber thus has the result of preserving laminar
flow and preventing bubbles from being entrapped.
[0066] According to this second method, valve 65 is electrically
actuated so as to transiently connect to the source of pressure.
The source of pressure is most conveniently a pressure pump
attached to a regulator. As soon as the reagent has filled the
chamber and emerged from the other fluid port 21, valve 65 is
switched back. An alternative method for providing such a short,
defined burst of pressure could be provided by a small syringe (not
shown). The syringe plunger could be connected to an electrical
actuator that rapidly drives it downwards until the plunger
traverses to the bottom of the chamber. The outlet of the syringe
could represent a source of a burst of pressure.
[0067] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
scope of the invention encompassed by the appended claims. For
example, although valves in the assembly are particularly effective
in holding the reagent in the wells, a very small hole might retain
the liquid due to capillary action.
* * * * *