U.S. patent application number 12/574468 was filed with the patent office on 2010-05-06 for apparatus and methods for efficient processing of biological samples on slides.
This patent application is currently assigned to AMERICAN REGISTRY OF PATHOLOGY. Invention is credited to Wei-Sing CHU.
Application Number | 20100112577 12/574468 |
Document ID | / |
Family ID | 22819279 |
Filed Date | 2010-05-06 |
United States Patent
Application |
20100112577 |
Kind Code |
A1 |
CHU; Wei-Sing |
May 6, 2010 |
APPARATUS AND METHODS FOR EFFICIENT PROCESSING OF BIOLOGICAL
SAMPLES ON SLIDES
Abstract
Methods for treating biological samples on microscope slides are
set forth. One aspect of the invention is the use of predried
reagents in wells on trays onto which the slides are placed,
especially the use of predried reagents which dissolve
sequentially. Yet another aspect of the invention is the use of
external controls placed directly on a microscope slide in
conjunction with a biological sample to be assayed. The external
controls can be conveniently placed on a membrane which can be
affixed to the slide. A further aspect of the invention is a
specially designed tray to allow whole chromosome painting of all
chromosomes of a cell sample on a single slide. The invention is
also drawn to a coverslip with concave wells which act as reaction
chambers when placed against a slide and filled with buffer.
Preferably a reagent is predried in the well. A further aspect of
the invention is a method of reacting samples on slides by placing
them into a reaction chamber together with a coverslip which has a
predried reagent on it.
Inventors: |
CHU; Wei-Sing; (Silver
Spring, MD) |
Correspondence
Address: |
ROTHWELL, FIGG, ERNST & MANBECK, P.C.
1425 K STREET, N.W., SUITE 800
WASHINGTON
DC
20005
US
|
Assignee: |
AMERICAN REGISTRY OF
PATHOLOGY
Washington
DC
|
Family ID: |
22819279 |
Appl. No.: |
12/574468 |
Filed: |
October 6, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11157922 |
Jun 22, 2005 |
7598036 |
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12574468 |
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09869082 |
Sep 24, 2001 |
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PCT/US99/30519 |
Dec 22, 1999 |
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11157922 |
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09219443 |
Dec 23, 1998 |
6703247 |
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09869082 |
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Current U.S.
Class: |
435/6.11 ;
422/400; 435/29; 435/6.12; 436/86; 436/94 |
Current CPC
Class: |
G01N 1/312 20130101;
Y10T 436/143333 20150115 |
Class at
Publication: |
435/6 ; 422/99;
435/29; 436/94; 436/86 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; B01L 3/00 20060101 B01L003/00; C12Q 1/02 20060101
C12Q001/02; G01N 33/48 20060101 G01N033/48 |
Claims
1-42. (canceled)
43. A multichamber coverslip comprising a group of connected
coverslips which are spaced such that a single coverslip lines up
with one or more slides in a slide holder of a system for
processing biological samples.
44. The multichamber coverslip of claim 43, wherein an individual
coverslip comprises a concave well.
45. The multichamber coverslip of claim 43, further comprising
glass.
46. The multichamber coverslip of claim 43, further comprising
plastic.
47. The multichamber coverslip of claim 44, wherein an individual
coverslip with a concave well is an incubation chamber.
48. The multichamber coverslip of claim 44, wherein the concave
well comprises a soft and pliable top.
49. The multichamber coverslip of claim 48, wherein the soft,
pliable top allows expansion and contraction.
50. The multichamber coverslip of claim 47 wherein the incubation
chamber comprises a volume between 10 and 20 .mu.l.
51. The multichamber coverslip of claim 43, wherein an individual
coverslip is prescored.
52. The multichamber coverslip of claim 49, wherein the prescored
coverslip can be snapped off the holder.
53. The multichamber coverslip of claim 43, further comprising
reagents dried thereupon.
54. The multichamber coverslip of claim 43, wherein each individual
coverslip is labeled.
55. The multichamber coverslip of claim 54, wherein the label is a
barcode.
56. A method of performing an assay on a biological sample
comprising, placing a microscope slide having a biological sample
into a slide holder of an integrated system for processing the
biological samples, wherein the system further comprises a
multichamber coverslip comprising a group of individual coverslips
which are connected and spaced such that a single coverslip is
lined up over the sample on a slide.
57. The method of claim 56, wherein a solution for an assay is
applied to the system.
58. The method of claim 57, wherein solution is water and each
coverslip further comprises a reagent predried onto the
coverslip.
59. The method of claim 56, wherein the assay is an
immunocytochemical assay, an in situ hybridization assay or a
polymerase chain reaction.
60. The method of claim 56 wherein the biological sample is
selected from the group consisting of a tissue section, a biopsy
tissue, a cell smear, a nucleic acid, a protein or peptide, a
chromosome, and a bodily fluid.
61. The method of claim 56, wherein the biological sample to be
assayed further comprises a control sample.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of co-pending U.S. patent
application Ser. No. 11/157,922, filed 22 Jun. 2005, which in turn
is a continuation of Ser. No. 09/869,082, filed 24 Sep. 2001, which
was filed under 35 U.S.C. .sctn.371 based on PCT/US99/30519, filed
22 Dec. 1999, which in turn is a continuation-in-part of U.S. Ser.
No. 09/219,443, filed 23 Dec. 1998, now U.S. Pat. No. 6,703,247.
Each application is incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] This invention relates to an apparatus for processing
biological samples on slides for a wide variety of purposes.
Biological samples are analyzed for many purposes using a variety
of different assays. Pathologists often use histochemistry or
immunocytochemistry for analyzing biological samples, molecular
biologists may perform in situ hybridization or in situ polymerase
chain reactions on biological samples, etc. Often the sample to be
analyzed will be embedded in paraffin and mounted on a microscope
slide.
[0003] The assays usually involve the use of antibodies, enzymes
and other expensive reagents and it is desirable to keep reagent
volume use to a minimum to lower costs. These assays are also quite
labor intensive although there are now some automated systems
(e.g., the Ventana ESIHC Staining System, the Shandon Lipshaw
Cadenza Automated Immunostainer; also see Brigati et al. (1988)).
The publications and other materials used herein to illuminate the
background of the invention or provide additional details
respecting the practice, are incorporated by reference, and for
convenience are respectively grouped in the appended List of
References. Most automated systems can only perform 40 to 48 slides
per run. Fisher automated systems can perform 120 slides per run.
Most automated systems which only perform immunocytochemistry do
not perform deparaffinizing, histochemistry (such as hematoxylin
and eosin staining) and coverslipping steps and these consequently
must be done separately by hand which is time and labor intensive.
The automated systems perform only a small part of the overall
process of preparing and analyzing slides. Steps which are still
manually performed prior to the automated portion include sorting
of cases and slides, labeling slides, programming the automated
equipment, daily antibody and reagent preparation, preparing
control tissue which is mounted on slides, and microwave antigen
retrieval. Procedures still performed manually after the automated
steps are dehydration, coverslipping, slide labeling and sorting of
slides and cases. Furthermore, most commercial ready-to-use
reagents are not suitable for automated systems which are required
to use specially designed reagents. Laboratories which process
large numbers of samples are likely to be willing to pay the high
cost associated with buying these automated systems as well as the
high cost of using the disposable accessories and reagents to
perform the assays, but small to intermediate sized laboratories
find it more cost effective to continue to process samples
manually.
[0004] A typical immunocytochemistry assay requires a series of
many steps. These include: obtaining a biological sample such as
from a biopsy, fixing the sample in formalin, processing the sample
overnight, embedding the sample in paraffin, cutting serial
sections and mounting on microscope slides and drying. These steps
are followed by steps to deparaffinize (treatments in xylene,
ethanol and water), and finally the reaction can be performed on
the sample which has been mounted on the slide. Typically a series
of solutions including reagents such as enzymes, primary antibody,
secondary antibody, detection reagent, chromogen, counterstain,
etc. is dropped onto the slide, incubated, and washed off. Finally
the sample may be viewed under the microscope. Clearly there are
many individual steps involved and each sample on a slide must be
processed individually. Besides being very labor intensive, there
are drawbacks associated with the commonly used method of simply
dropping solutions on top of the mounted sample on the microscope
slide. The solution is not restricted simply to the area of the
biological sample itself and the solution may be relatively deep
rather than being a thin layer. These features require use of extra
reagents which are quite expensive. Leaving the solutions open to
the air as they sit on the slide also may lead to evaporation if
the samples must incubate for a long period of time. Evaporation
leads to concentration or drying out of the reagents and high
concentrations may lead to increased background levels which are
clearly undesirable. If the solutions evaporate totally the assay
will fail. Incubating samples in humidity chambers with covers may
prevent evaporation problems, but water droplets which condense
onto the humidity chamber cover may fall onto the slides and this
will ruin the assay.
[0005] Improved methods for more rapidly assaying several samples
at once, but without the high cost of automated systems, will be
welcomed by small to intermediate sized laboratories. Furthermore,
methods which will allow use of smaller amounts of reagents and
overcome the drawbacks of processing samples on slides open to the
atmosphere will be a welcome advance.
SUMMARY OF THE INVENTION
[0006] The present invention relates to an apparati and methods for
performing assays on biological samples mounted on microscope
slides. Use of the apparati and/or methods aid in making assays
more rapid and convenient. One aspect of the invention is the use
of reagents which are predried in the wells of the tray thereby
simply necessitating the addition of water or buffer to the well
without having to add the reagents at the time of assay. The well
is then covered with a slide with a biological sample premounted on
the slide. The different wells of a multiwell tray can be
pretreated with different reagents dried in each well. Multistep
assays can be performed by moving a slideholder with attached
slides from one multiwell tray to the next, with each well of a
multiwell tray having the desired reagents predried on it. A
variation of this is to employ a multilayer coating of reagents in
each well such that the first set of reagents dissolves quickly and
acts upon the biological sample, the second layer then dissolves
releasing the reagents for the second step, etc., thereby requiring
the use of fewer trays, possibly only a single tray.
[0007] Another aspect of the invention is to have built in controls
on each slide. This is a portion of the slide to which are attached
positive and negative controls. These controls allow one to
determine whether the assay has worked properly for each individual
slide since each slide has its own set of controls and which
simultaneously act as labels for each slide.
[0008] The invention is also directed to a coverslip with concave
wells for holding reagents. The coverslip can be mounted onto a
slide so that it will hold reagents for performing analyses but is
easily removed to allow washing of the slide. The cover slip can
include controls dried onto it for the assay.
[0009] Another aspect of the invention is automated processing of
biological samples in a reaction chamber in conjunction with a
coverslip which has reagents predried onto it and can optionally
have control sample prespotted onto it.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1 illustrates a slideholder 1 with slides 70.
Slideholder 1 includes a handle 5 with holes 11. Openings 7 in the
slideholder allow labels on slides 70 to be seen. Labels may also
be attached directly to the slideholder 1 at region 15. Slides 70
are inserted into slots 56 of slideholder 1.
[0011] FIGS. 2A-B illustrate a tray 14 and slides 70. FIG. 2A is a
front elevational view of tray 14. Wells 24 are separated by
troughs 38. Boundaries 44 of wells 24 are flat and are elevated
above the interior portion of the wells 24. Trough 90 is contiguous
with troughs 38. FIG. 2B is a cross sectional view of tray 14 taken
along line 54-54 of FIG. 2A. This view shows wells 24, troughs 38,
and well boundaries 44. Slide 70 is shown resting on one well
24.
[0012] FIG. 3 illustrates a slide 70 with a biological sample 220
and a stamp 230. The stamp shown contains reagents A-F.
[0013] FIG. 4 illustrates a well 24 in which three reagents
(indicated as 250, 260, and 270) have been dried and onto which has
been placed a slide 70 with mounted biological sample 220. Layers
of inert material separating the layers of reagents from each other
are not shown.
[0014] FIGS. 5A-B illustrate one well of a multiwell tray 330 which
is used to automate several steps of the procedure of assaying a
biological sample in conjunction with a thermal cycler, pumps and a
central processing unit. FIG. 5A shows slide 70 with mounted
biological sample 220 placed on a well or reaction chamber 280.
Inlets 300 and 302 and outlets 294 and 296 which connect to
reaction chamber 280 are illustrated. The portion of tray 330 which
forms the bottom of the reaction chamber 280 is shown as 282.
Optional stops 281 are shown which prevent the reaction chamber
bottom 282 from pressing up against sample 220. The view in FIG. 5A
shows the reaction chamber bottom 282 in an "open" mode which
causes the reaction chamber 280 to have a large volume. FIG. 5B
shows the tray and slide of FIG. 5A in conjunction with other
optional equipment. In FIG. 5B the reaction chamber bottom 282 is
in a "closed" mode such that reaction chamber 280 encompasses a
smaller volume than seen in FIG. 5A. Piston 284 to move reaction
chamber bottom 282 is shown. The piston 284 is controlled by
central processing unit 286. A thermal cycler 288 is illustrated
pressed against slide 70. The thermal cycler can also be controlled
by central processing unit 286. Tubing can be attached to the
inlets 300 and 302 and to the outlets 294 and 296. Pumps 290
attached to the tubing are shown and pump liquid to or from
reservoirs 291 or 292 or to gel 298.
[0015] FIGS. 6A-E illustrate a tray used to perform whole
chromosome painting of multiple chromosomes on cells on a single
slide or which can be used to perform in situ hybridization or FISH
on a biological sample. FIG. 6A illustrates an 8 well tray 400 with
wells 410. Each well is separated from neighboring wells by troughs
420. Each well 410 has an opening or channel 430 through which
liquid can be pipetted. FIG. 6B is a side view of the 8 well tray
400 shown in FIG. 6A. A slide 70 is shown on the tray 400. Four
wells 410 are illustrated with three of the wells being empty and
one shown filled with liquid. Openings 430 and troughs 420 are also
illustrated. FIG. 6C is an end-on view of the slide and tray of
FIGS. 6A and 6B. Trough 420 is shown between two wells 410.
Openings 430 into the wells 410 are shown. Slide 70 is shown
resting above sides of tray 400 showing optional clips 402 to hold
slide 70 to tray 400. FIG. 6D is a schematic showing a slide 70
illustrating 8 regions 440 of the slide which will be in contact
with each of the 8 wells 410. This is only illustrative, there
being no need to actually denote these regions 440 on the slides
used in practice. FIG. 6E illustrates one manner of designing
built-in controls on slide 70 by showing an enlargement of one
region 440. Each region 440 has nucleic acids 442, which hybridize
to the probes being used in the assay, placed in an array around
the perimeter of region 440. These controls will be in contact with
probe during the hybridization.
[0016] FIGS. 7A-H illustrate the processing of a biological sample
on a slide in conjunction with a coverslip with concave wells.
[0017] FIGS. 8A-D illustrate processing of a biological sample on a
microscope slide in conjunction with a coverslip.
DETAILED DESCRIPTION OF THE INVENTION
[0018] The present invention is an integrated system for processing
biological samples on microscope slides in a more rapid and
efficient and less costly manner than is typical. Much of the
background for this disclosure is shown in U.S. Pat. No. 5,958,341
(W.-S. Chu; issued Sep. 28, 1999) which is incorporated herein by
reference. The numbering of parts used in this disclosure, if not
shown in a Figure herein, refers to the numbering shown in Figures
of U.S. Pat. No. 5,958,341 (W.-S. Chu).
[0019] By a biological sample is meant a tissue section, biopsy,
cell smear, nucleic acid, protein or peptide, chromosome, bodily
fluid or other biological material commonly observed under a
microscope. The system as illustrated in FIGS. 1 and 2A-B consists
of a slideholder and a tray or a coverslip (see FIGS. 7A-H and
8A-D) for simultaneously holding multiple, preferably up to six,
microscope slides to allow for concurrent processing of the
multiple slides. The slideholder may be reusable.
[0020] In practice, a biological sample is mounted onto each of the
slides to be analyzed. This often involves steps of fixing a
biological sample in formalin, embedding the sample in paraffin,
cutting thin, serial sections from the paraffin or from frozen
tissue sections and mounting the sections onto the microscope
slides. These are dried overnight at room temperature. The mounted
biological samples are subjected to some type of assay such as
staining. For this the mounted samples must be placed in contact
with a series of solutions with washing steps in between each
different change of reagent. In the present invention the reagents
are measured into or predried in each well 24 in the trays 14.
Enough reagent or buffer is added to completely fill the well 24
such that the solution in the well 24 will contact the microscope
slide 70 which is to be laid on top of the well 24. There should be
no air bubbles present between the solution in the well 24 and the
microscope slide 70. By exactly filling the well 24 or by slightly
overfilling the well 24 so that there is a slight overflow once the
slide 70 is placed on top of the well 24 (surface tension holding
the top of the solution in the well 24 prior to a slide 70 being
placed onto it) there is no problem with air bubbles forming.
Capillary action of the fluid in the well 24 contacting the slide
70 allows for good contact between the biological sample and
reagents across the complete well 24 area and helps to seal the
well 24. Trays 14 may be designed to include a hook on one edge of
a well boundary 44. This is shown in FIGS. 4C and 4F of U.S. Pat.
No. 5,958,341 (W.-S. Chu). By pushing all of the slides 70 against
the hooks, all of the slides will be held against the well
boundaries 44 and this will assure good contact with the reagents
within the wells 24.
[0021] By placing the slides 70 onto the tray 14 in the above
manner, the mounted biological sample is facing down into the well
24 and is not exposed to the atmosphere. This prevents extraneous
material from falling into the reagent or onto the biological
sample during incubation. Furthermore, the slide 70 covers the well
24 and helps to prevent evaporation of the reagent solution in the
well 24 during incubation. Evaporation may lead to very bad
background signals. The present invention helps to overcome this
problem.
[0022] After incubation with each reagent the slideholder 1 and
tray 14 are picked up and put into a standard staining dish with
500 milliliters of phosphate buffered saline (PBS) solution. Once
in the PBS, the surface tension between the slides 70 and the tray
14 disappears and the slides are very easily removed from the tray.
The slides are then put through the appropriate washing steps. It
is a simple matter to pick up six slides 70 at once since they are
all attached to a single holder 1. A standard staining dish in a
laboratory is large enough to accommodate six slides 70 across (as
attached to a single slideholder 1) and can contain 20 slideholders
1. Therefore 120 slides 70 may be washed and processed
simultaneously.
[0023] The above methods are an improvement because they result in
an enclosed assay system which helps to prevent contamination.
Also, the enclosed system prevents evaporation resulting in a
constant volume of reagent being present thereby resulting in a
known amount of and constant concentration of reagents. These
features lead to better and more consistent results than prior art
methods, e.g., those wherein reagents are simply dropped on top of
a tissue sample mounted on a slide and which is open to the
atmosphere thereby allowing contamination and evaporation.
[0024] Another aspect of the invention is to predry reagents in
wells 24 of trays 14 thereby requiring simply the immersion of the
tray 14 and slides 70 into water or buffer or the pipetting of
water or a buffer into the wells 24 at the time of assay. Trays 14
can be prepared which include a series of reagents predried in the
wells 24 of a multiwell tray 14, e.g., each well 24 of a multiwell
tray 14 can have a different set of reagents dried in the well 24.
At the time of assay, slides 70 can have a biological sample from a
single patient or from different patients mounted on them and be
placed onto a single tray 14 to perform multiple assays at once.
Such trays 14 with predried reagents can be prepared ahead of time
and stored until the time of use. As currently practiced, assays
performed on biological samples are performed by fixing a sample
onto a slide and then dropping reagents onto the sample. Such a
method cannot take advantage of premeasured, predried reagents
which require only the addition of water or buffer. In the
invention disclosed here, the reagents can be predried in a well 24
on a tray 14, buffer or water is added to well 24, and a slide 70
with biological sample mounted on it is placed on top of well 24,
sample side down. The buffer or water may be added to well 24 via
tubing after placing slide 70 on top of well 24. Having slide 70
over well 24 forms a sealed reaction chamber which prevents
contamination and evaporation and also ensures uniform distribution
of reagents as compared to dropping solution on top of a slide as
is generally done in current practice.
[0025] Yet another aspect of the invention is to have built-in
controls and/or labels on each slide. Known controls are
immobilized onto each slide in a region apart from the biological
sample. For example, the controls can be antigens, peptides,
proteins or cells which are being tested for in the biological
sample or can be a nucleic acid of known sequence if a
hybridization assay is being performed. These would act as positive
controls which should give a signal or color if the assay works
properly. Negative controls can also be placed onto the slide,
e.g., a protein or antigen or a nucleic acid which should not react
with the reagents in the well. For example, assume a person is to
be tested for the presence of six antigenic determinants A-F. A six
well tray can be used with each well containing a different
antibody A'-F'. The six different antigenic determinants can be
spotted onto all six slides. In all cases, only a single one of
these controls should show as positive on each slide. Slide A
should show only antigenic determinant A as a positive signal,
slide B should show only antigenic determinant B as a positive
signal, etc. These act as external controls. If more than one
control shows as a positive, this indicates antibody cross reaction
has occurred. If none of the controls is positive it indicates that
the reaction did not work, e.g., a reagent may have been missing.
The biological sample being tested acts as an internal control.
[0026] The external controls can be placed onto each slide by a
variety of means. A preferred mode is to spot the reagents onto the
equivalent of a postage stamp or sticker, which uses glue resistant
to xylene and alcohol, which can then be glued onto each slide.
Such a stamp or sticker can be made of any suitable material to
which proteins, peptides, cells or nucleic acids bind tightly. This
can include, but is not limited to, commonly used membranes such as
nitrocellulose, plastic, glass or nylon. Specific examples of such
membranous material are nitrocellulose itself, Immobilon-P
(Millipore), Hybond-N, Hybond-N.sup.+ and Hybond C-extra
nitrocellulose (Amersham), Genescreen and Genescreen Plus (Du
Pont), Clearblot-P (ATTO Co.) and polyvinyldifluoride membranes
(Millipore or BioRad). The stamp or sticker will have regions A-F
as shown in FIG. 3. These stamps or stickers can be premanufactured
and stored until ready for use, the antigenic determinants,
proteins, peptides, cells or nucleic acids being dried onto the
stamps or stickers. The name of the antigen, protein, cell, etc.,
can be printed on the stamp or sticker. This is especially suitable
for mass production. Standard sets of assays can be premade such as
a panel to test for breast cancer or a panel to test for Hodgkin's
disease, but one can always design any combination of reagents as
external controls as are desired. A stamp of controls can be
attached to a slide either prior to a biological sample being
placed upon the slide or it may be delayed until the biological
sample has been fixed on a slide and been processed to the point at
which reactions relevant to the controls are to be performed.
[0027] The stamps can be color coded or numbered to indicate a
specific panel of tests to be performed. In like fashion the tray
14 can be color coded or numbered or otherwise marked to indicate
the panel of tests to be performed, this being dependent upon the
predried reagents in the wells 24 of the tray 14. The stamp and the
tray should match colors or numbers or other marking.
[0028] One other aspect of the invention is that reagents which are
dried in wells 24 can be dried in layers in the reverse order which
they are to act. When buffer is added the last added reagent will
dissolve first and be active, followed by the next to last added
reagent which acts in turn, etc. In this manner two or more
reagents can be added to a single well 24 thereby allowing
consecutive action of the reagents without the necessity of moving
the slides 70 from one tray 14 to a second tray 14. For multistep
reactions this will decrease the number of trays 14 which are
necessary and also decreases the amount of labor involved.
[0029] Another aspect of the invention is a specially designed tray
or chip which allows one to perform whole chromosome painting of
all 24 human chromosomes on cells on a single slide.
[0030] Still another aspect of the invention is a tray and slide
assembly wherein the volume of space in the well of the tray can be
adjusted so that a small volume can be present to perform a
reaction such as a PCR and then the volume of space can be
increased to allow fluid to be pumped through the well.
[0031] Those of skill in the art recognize that the sample to be
tested on the slide including the protein, peptide, DNA, RNA or
cells or the control protein, peptide, DNA, RNA or cells on the
stamp, must be immobilized so that they will not be released during
the assay. The reagents which may have been predried in the tray,
however, which reagents may include proteins, peptides, nucleic
acids, etc., should be released, in a programmed order if
multilayered, once the water or buffer has been added.
EXAMPLES
[0032] In each example a biological sample is first mounted onto a
microscope slide 70 and then assayed. Surgical and autopsy human
biological samples from various organs (lymph node, liver, kidney,
lung, breast, skin, prostate) were routinely fixed in 10% neutral
buffered formalin, processed overnight on a tissue processor, and
embedded in paraffin. Serial sections are cut at 4-5 microns and
mounted onto Probe-On-Plus Slides (#15-188-52; Fisher Scientific)
and dried overnight at room temperature. Slides 70 are then
inserted into a reusable slideholder 1. At this point all the
slides 70 in a single holder 1 (up to six slides) can be handled
simultaneously. The slides 70 are deparaffinized by placing the
slides 70 in a staining dish with four changes of xylene for 5
minutes each, two treatments of 100% ethanol for 1 minute each and
two treatments of 95% ethanol for 1 minute each. The deparaffinized
tissue section slides 70 are cleared and washed with deionized
water.
[0033] The present invention is further detailed in the following
Examples, which are offered by way of illustration and are not
intended to limit the invention in any manner. Standard techniques
well known in the art or the techniques specifically described
below are utilized.
Example 1
Immunocytochemistry
[0034] In this Example a biological sample is treated with
antibodies (primary and secondary), treated for chromogen color
development, and finally counterstained.
A. Proteolytic Pretreatment of Mounted Tissue Samples
[0035] It is well known in the art that when using certain
antibodies for immunocytochemical staining it is necessary to
pretreat the formalin fixed tissue section with proteolytic enzymes
such as 0.4% pepsin, pH 2.0. When this is necessary the following
steps may be utilized. A few drops (150-200 .mu.L) of the
proteolytic digestion solution are placed on each well 24 of the 3
or 6 well tray 14. The tissue side of the slides 70 is faced down
on the wells 24. The slideholder 1 with the slides 70 should be
slowly laid down and placed on the wells 24 of the tray 14. No air
bubbles should remain between the tissue side of the slides 70 and
the solution in the wells 24 of the tray 14. The slides 70,
slideholder 1 and tray 14 with solution are incubated for 15
minutes at 40.degree. C.
[0036] If many samples are being processed at one time it is more
efficient to forgo use of the tray 14 during this proteolytic
pretreatment step. The slides 70 are still placed into slideholders
1 six to a holder 1. The slideholders 1 and slides 70 are then
placed vertically into a staining dish with 500 mL of the
proteolytic digestion solution (which may be reused) and incubated
for 20 minutes at 40.degree. C. in a water bath. Up to twenty
slideholders 1 (120 slides) may be simultaneously placed into the
staining dish for this pretreatment step.
[0037] Some antibodies require that the tissue section be
pretreated with microwave antigen retrieval. Slideholders 1 (up to
20) with slides 70 are vertically placed into a staining dish with
500 mL of 0.01 M citrate buffer, the staining dish is placed in the
center of a microwave oven, and the oven is turned to high power
(800-850 Watts) for 7-8 minutes bringing the solution to a rapid
boil. The oven is turned off, the power level is reset to 400
Watts, and the oven is turned on again to heat the solution for 7-8
minutes.
[0038] After proteolytic digestion and microwave treatment the
tissue sections are washed in the staining dish with three 500 mL
changes of phosphate buffered saline (PBS).
B. Treatment of Tissue Sections with Goat and Horse Serum
[0039] All slides 70, whether or not proteolytically digested and
microwave treated, are incubated with 5% mixed normal goat and
horse serum for 20-30 minutes at room temperature. Each well 24 of
a tray 14 is filled (approximately 150-200 .mu.L) with mixed normal
goat and horse serum. The tissue side of the slides 70 is placed
down on the wells 24 to contact the serum. The slideholder 1 should
be slowly laid down so as to avoid trapping any air between the
slides 70 and the wells 24. Again, if many samples are being
processed at one time, it is more efficient to perform this step as
a batch by placing up to 20 slideholders 1 vertically into a
staining dish with 500 mL of 5% mixed normal goat and horse serum
for 20-30 minutes.
C. Application of the Primary Antisera or Antibodies
[0040] Following incubation with the serum, the slideholder 1 and
slides 70 as well as the tray 14 are put into a staining dish with
PBS. The tray 14 is separated from the slideholder 1 and both are
washed once with PBS. The washed tray 14 may be reused for the next
step. Prediluted primary antisera or antibodies (approximately
150-200 .mu.L) are applied to each well 24 of the tray 14. The
washed slides 70, still in the slideholder 1, are placed tissue
side down onto the wells 24. As always care must be taken to avoid
trapping bubbles between the slide 70 and the reagent solution in
the wells 24. The samples are incubated with the antisera or
antibodies for 2-4 hours at room temperature or incubated in a
humidity chamber at 40.degree. C. for 2 hours or may be incubated
in a humidity chamber at room temperature overnight. After
incubation the slideholder 1 and attached slides 70 are removed
from the tray 14 and are washed in a staining dish with PBS three
times.
D. Application of the Secondary Antibody
[0041] Prediluted secondary antibody (approximately 150-200 .mu.L)
is applied into each well 24 of a new tray 14. The slides 70 in the
slideholder 1 are placed onto the wells 24 tissue side down being
careful to avoid bubbles. This is incubated for 30 minutes at
40.degree. C. in a humidity chamber. After incubation the
slideholders 1 and attached slides 70 are removed from the tray 14
and washed in a staining dish with three changes of PBS.
E. Treatment for Removal of Endogenous Peroxidase Activity
[0042] All slideholders 1 with attached slides 70 are placed into a
staining dish with 500 mL of PBS with 3% hydrogen peroxide and 0.1%
sodium azide, and incubated at room temperature for 15 minutes.
After incubation with the hydrogen peroxide PBS the slideholders 1
and attached slides 70 are washed in a staining dish with three
changes of PBS.
F. Application of the ABC Complex "ELITE"
[0043] The ABC complex (Vector Laboratories Inc., Burlingame,
Calif.) is diluted to its working concentration using PBS. The
working concentration (approximately 150-200 .mu.L) is applied to
each well 24 of a new tray 14. The slides 70 with attached
slideholders 1 are carefully placed tissue side down onto the trays
14 so that no air bubbles are trapped between the solution and the
slides 70. The slides 70 and trays 14 with ABC solution are
incubated in the humidity chamber at 40.degree. C. for 30 minutes.
After incubation the slideholders 1 with attached slides 70 are
removed from the trays 14 and washed in a staining dish with 3
changes of PBS.
G. Chromogen Color Development Using Diaminobenzidine (DAB)
[0044] DAB solution is prepared by adding 100 mg DAB to 100 mL PBS
and adding 50 .mu.L of 30% H.sub.2O.sub.2. Approximately 150-200
.mu.L of the DAB solution is added to each well 24 of a new tray 14
to completely fill each well 24. The slides 70 with attached
slideholders 1 are placed tissue side down onto the wells 24 being
careful to avoid trapping air bubbles. Color development can be
monitored by viewing the slideholders 1 and trays 14 with DAB under
a microscope. A colored precipitate will form at the site of
positive cells. Color begins to appear after 2-5 minutes, usually
reaching sufficient development within 10 minutes, but a 20-30
minute incubation may be necessary for weakly stained samples. To
stop development, all slideholders 1 with slides 70 are removed
from the trays 14 and washed in a staining dish with three changes
of deionized water.
H. Counterstaining
[0045] Slideholders 1 and attached slides 70 are immersed in
Harris's hematoxylin for 10-50 seconds and washed by dipping into
deionized water for three changes. Then all the slides 70 are
immersed in 0.2% ammonium hydroxide solution for 30 seconds and
washed by dipping in deionized water for 3 changes. The slides 70
are dipped into 95% ethanol for two changes of 2 minutes each,
followed by dipping into 100% ethanol for 2 changes of 2 minutes
each, and finally the slides 70 are cleared by dipping into two
changes of xylene for 2 minutes each.
I. Attachment of the Coverslip
[0046] Place 1 drop of Cytoseal 60 or premount on the tissue
section side of each slide 70 with the slides 70 still attached to
the slideholder 1. Place coverslips onto each slide 70. Although
this may be done one by one, it is more efficient to use a
specially designed coverslip which is actually six (or three)
conjoined coverslips properly spaced to align with six (or three)
slides 70. Using this special coverslip, up to 6 individual
coverslips are effectively aligned and placed onto slides 70
simultaneously. The coverslips are easily separated from the
plastic strip holding them together simply by bending the coverslip
which is prescored to allow the strip to snap apart from the
coverslips which remain bound to the slides 70. At this point the
slides 70 may be removed from the slideholder 1 to be handled
individually, or they may be left attached to the slideholder 1 for
ease of transportation.
[0047] FIGS. 10-12 of U.S. Pat. No. 5,958,341 (W.-S. Chu) show the
results of a study comparing the use of the present invention with
staining methods simply using the standard manual method of
dropping reagents onto the surface of a slide-mounted tissue sample
and leaving the reagents open to the atmosphere for incubation. The
Figures show that the results obtained with the two methods are
extremely comparable with the results obtained using the present
invention being at least as good as, and apparently better than,
the results obtained using the traditional method. The present
invention however allowed these results to be obtained with less
work and with the use of smaller amounts of reagents.
[0048] Comparing the two methods, the background staining is
significantly reduced by using the present invention, especially
when using polyclonal antibodies (anti-kappa light chain antibodies
and anti-lambda light chain antibodies). The invention
significantly improves the staining results by reducing the
background. Background is partially due to free FC fragments which
precipitate by gravity and bind nonspecifically to the tissue. The
present method inverts the slide such that the tissue is above the
solution and therefore free FC fragments cannot precipitate by
gravity onto the tissue.
Example 2
In Situ Hybridization
[0049] In this example biological samples are mounted onto slides
70, hybridized with biotin or digoxigenin labeled probes and
reacted with anti-biotin or anti-digoxigenin antibody. The samples
are then stained.
A. Preparation and Mounting of Tissue Sample
[0050] A tissue sample is prepared as described above but with
extra measures to prevent nucleic acid degradation. A tissue sample
is fixed in 10% neutral buffered formalin, processed overnight on a
tissue processor, embedded in paraffin, cut into serial sections of
4-5 microns, mounted onto Probe-On-Plus Slides (#15-188-52; Fisher
Scientific), and dried overnight at room temperature. The slides 70
are inserted into a slideholder 1 and are deparaffinized by placing
into a staining dish. The slides 70 are treated with four changes
of xylene for 5 minutes each, two changes of 100% ethanol for 1
minute each and two changes of 95% ethanol for 1 minute each. The
deparaffinized tissue section slides are then cleared and washed
with deionized water with RNase Block (BioGenex, San Ramon,
Calif.).
B. Proteinase K Treatment of the Mounted Tissue Samples
[0051] Approximately 150-200 .mu.L of freshly diluted proteinase K
solution is placed into each well 24 of a tray 14 to completely
fill each well 24. The microscope slides 70 (still in the
slideholder 1) are placed onto the wells 24 with the tissue side
down. The slides 70 are placed onto the wells 24 carefully so as to
avoid the presence of air bubbles between the solution in the wells
24 and the slide 70. This is incubated for 15 minutes at room
temperature.
[0052] After digestion, the slideholders 1 with slides 70 attached
are removed from the tray 14 and washed in a staining dish with 500
mL of PBS with RNase Block for 5 minutes. The tissue section slides
70 are dehydrated by immersing in a staining dish serially in the
following solutions: 500 mL distilled water plus RNase Block for 10
seconds, 500 mL 50% ethanol plus RNase Block for 10 seconds, 500 mL
of 95% ethanol for 10 seconds, and 500 mL 100% ethanol for 10
seconds. The slides 70 are dried at room temperature for 5
minutes.
C. Hybridization with Biotinylated or Digoxigenin Labeled
Probes
[0053] Trays 14 with shallow wells 24 (0.02-0.08 mm in depth) may
be used here to conserve materials. Hybridization solution
containing a biotinylated or digoxigenin labeled oligonucleotide
probe is placed into each well 24 of a tray 14. Enough solution is
added to each well 24 to completely fill the well 24. This requires
approximately 50-100 .mu.L of solution. The slides 70 are placed on
top of the wells 24 (3 or 6 at a time still attached to the
slideholders 1) being careful not to trap any air bubbles. The
trays 14 plus slideholders 1 and slides 70 are placed in an oven or
on a heating block at 95.degree. C. for 8-10 minutes to denature
the nucleic acids. This step eliminates hair-pin loops or folding
back of mRNA sequences. After the denaturation step, the slides 70
are incubated in a humidity chamber at 45.degree. C. overnight.
Following the hybridization step, the slides 70 are washed by
removing the slideholders 1 with attached slides 70 from the trays
14 and washing the slides 70 in a staining dish with 2.times.SSC
(standard saline citrate) at 37.degree. C. for 5 minutes followed
by a wash with 1.times.SSC at 37.degree. C. for 5 minutes. This is
followed by a 30 minute wash in 0.2.times.SSC at 60.degree. C.
Finally the slides 70 are washed with 2 changes of PBS for 2-5
minutes each.
D. Signal Detection
[0054] The slideholders 1 with attached slides 70 are placed
vertically into a staining dish with 500 mL of 5% mixed normal goat
and horse serum at room temperature for 20 minutes. Prediluted
mouse anti-biotin or mouse anti-digoxigenin antibody (150-200
.mu.L) is applied to each well 24 of a new tray 14. The slides 70
are placed onto the wells 24 of the tray 14 taking care to avoid
trapping bubbles. The slides 70 and trays 14 with antibody are
incubated in a humidity chamber at 40.degree. C. for 2 hours.
[0055] After incubation with the anti-biotin or anti-digoxigenin
antibody, the slideholders 1 with slides 70 are removed from the
trays 14 and washed in a staining dish with three changes of
PBS.
E. Application of the Secondary Antibody
[0056] Prediluted secondary antibody (approximately 150-200 .mu.L)
is applied into each well 24 of a new tray 14. The slides 70 in the
slideholder 1 are placed onto the wells 24 tissue side down being
careful to avoid bubbles. This is incubated for 30 minutes at
40.degree. C. in a humidity chamber. After incubation the
slideholders 1 and attached slides 70 are removed from the tray 14
and washed in a staining dish with three changes of PBS.
F. Treatment for Removal of Endogenous Peroxidase Activity
[0057] All slideholders 1 with attached slides 70 are placed into a
staining dish with 500 mL of PBS with 3% hydrogen peroxide and 0.1%
sodium azide, and incubated at room temperature for 15 minutes.
After incubation with the hydrogen peroxide PBS the slideholders 1
and attached slides 70 are washed in a staining dish with three
changes of PBS.
G. Application of the ABC Complex "ELITE"
[0058] The ABC complex is diluted to its working concentration
using PBS. The working concentration (approximately 150-200 .mu.L)
is applied to each well 24 of a new tray 14. The slides 70 with
attached slideholders 1 are carefully placed tissue side down onto
the trays 14 so that no air bubbles are trapped between the
solution and the slides 70. The slides 70 and trays 14 with ABC
solution are incubated in the humidity chamber at 40.degree. C. for
30 minutes. After incubation the slideholders 1 with attached
slides 70 are removed from the trays 14 and washed in a staining
dish with 3 changes of PBS.
H. Chromogen Color Development Using Diaminobenzidine (DAB)
[0059] DAB solution is prepared by adding 100 mg DAB to 100 mL PBS
and adding 50 .mu.L of 30% H.sub.2O.sub.2. Approximately 150-200
.mu.L of the DAB solution is added to each well 24 of a new tray 14
to completely fill each well 24. The slides 70 with attached
slideholders 1 are placed tissue side down onto the wells 24 being
careful to avoid trapping air bubbles. Color development can be
monitored by viewing the slideholders 1 and trays 14 with DAB under
a microscope. A colored precipitate will form at the site of
positive cells. Color begins to appear after 2-5 minutes, usually
reaching sufficient development within 10 minutes, but a 20-30
minute incubation may be necessary for weakly stained samples. To
stop development, all slideholders 1 with slides 70 are removed
from the trays 14 and washed in a staining dish with three changes
of deionized water.
I. Counterstaining
[0060] Slideholders 1 and attached slides 70 are immersed in
Harris's hematoxylin for 10-50 seconds and washed by dipping into
deionized water for three changes. All the slides 70 are immersed
in 0.2% ammonium hydroxide solution for 30 seconds and washed by
dipping in deionized water for 3 changes. The slides 70 are then
dipped into 95% ethanol for two changes of 2 minutes each, followed
by dipping into 100% ethanol for 2 changes of 2 minutes each, and
finally the slides 70 are cleared by dipping into two changes of
xylene for 2 minutes each.
J. Coverslipping
[0061] Place 1 drop of Cytoseal 60 or premount on the tissue
section side of each slide 70 with the slides 70 still attached to
the slideholder 1. Place coverslips onto each slide 70. Although
this may be done one by one, it is more efficient to use a
specially designed coverslip which is actually six (or three)
conjoined coverslips properly spaced to all line up with six (or
three) slides 70. Using this special coverslip, up to 6 individual
coverslips are effectively aligned and placed onto slides 70
simultaneously. The coverslips are easily separated from the
plastic strip holding them together simply by bending the strip
which is prescored to allow the strip to snap apart from the
coverslips which remain bound to the slides 70. At this point the
slides 70 may be removed from the slideholder 1 to be handled
individually, or they may be left attached to the slideholder 1 for
ease of transportation.
Example 3
PCR In Situ Hybridization
[0062] Polymerase chain reaction (PCR) was developed as an in vitro
method for amplifying small amounts of specific pieces of nucleic
acids. This was later adapted to in situ studies so that there was
amplification of nucleic acid within tissue sections. The apparatus
of the present invention is suited to performing these in situ
PCRs. An example of a PCR in situ hybridization protocol is given
in Nuovo (1994).
A. In situ PCR
[0063] Serial tissue sections are cut at 4-5 microns thickness,
mounted onto Probe-On-Plus slides 70, and dried overnight at room
temperature. The mounted tissue sections are deparaffinized and
digested with pepsin at 40.degree. C. for 15-90 minutes depending
on the length of time of fixation in formalin. The pepsin is
inactivated by washing the slides 70 in diethylpyrocarbonate (DEPC)
treated water for one minute followed by a one minute wash in 100%
ethanol. The slides 70 are then air dried.
[0064] Polymerase chain reaction solutions are made according to
any standard procedure. See, e.g., K. B. Mullis et al., U.S. Pat.
No. 4,800,159. Combine buffer, 5' and 3' primers, water, Taq
polymerase (AmpliTaq, Perkin Elmer) (or other thermophilic
polymerase) and Self-Seal Reagent (MJ Research, Inc.) in a total
volume of 20-50 .mu.L. Apply the 20-50 .mu.L of solution to a well
24 of a specially designed in situ PCR aluminum tray 14. The trays
14 to be used in Example 1 are preferably made of a disposable
plastic material, but the trays 14 used for PCR studies must be
capable of being cycled through a series of temperatures which may
reach 95-100.degree. C. Therefore it is necessary for such trays 14
to be heat resistant (i.e., they should not melt or otherwise be
destroyed by high temperatures) and also to be good conductors of
heat. Aluminum is a preferred material from which to manufacture
these trays 14. These aluminum trays 14 have wells 24 which are
0.005-0.03 mm in depth and hold approximately 20-50 .mu.L of
solution.
[0065] After completely filling each well 24 of the aluminum tray
14, the slideholder 1 and attached slides 70 are placed on top of
the tray 14 with the tissue section facing down so as to contact
the solution in the well 24 upon which it is placed. Care must be
taken to avoid air bubbles being present between the solution and
the slide. The slideholder 1, slides 70 and aluminum tray 14 are
then placed onto a block of a thermal cycler at 95.degree. C. for
3-5 minutes to denature the nucleic acids in the tissue. Twenty to
thirty cycles are then performed cycling between 60.degree. C. for
2 minutes and 94.degree. C. for 1 minute.
[0066] Following the cycling steps, the slideholder 1, slides 70
and aluminum tray 14 are placed vertically into a staining dish
with 2.times.SSC at 37.degree. C. for 5 minutes. The slideholder 1
is removed from the aluminum tray 14 and washed with
0.5-1.times.SSC at 37-60.degree. C. for 10-30 minutes (depending
upon background). In situ hybridization is performed as described
in Example 2 using a biotinylated or digoxigenin labeled probe
chosen internal to the primers.
B. Reverse Transcriptase In Situ PCR
[0067] Serial tissue sections are cut at 4-5 microns thickness,
mounted onto Probe-On-Plus slides 70, and dried overnight at room
temperature. An important aspect of the RT in situ PCR is that both
negative and positive controls be performed and it is preferred
that these be performed on the same glass slide. The positive
control omits the DNAse digestion step and should generate an
intense nuclear signal from target specific amplification, DNA
repair and mispriming. The negative control uses a DNAse treatment
plus primers that do not correspond to a target in the cells. The
test sample undergoes DNAse treatment but uses primers specific to
the desired target nucleic acid. The mounted tissue sections are
deparaffinized and digested with pepsin at 40.degree. C. for 15-90
minutes depending on the length of time of fixation in formalin.
The pepsin is inactivated by washing the slides 70 in
diethylpyrocarbonate (DEPC) treated water for one minute followed
by a one minute wash in 100% ethanol. The slides 70 are then air
dried.
[0068] Digest two of the three mounted tissue sections with
RNase-free DNAse by filling each well 24 of a plastic tray 14
(requiring approximately 150-200 .mu.L) with prediluted RNase-free
DNAse and placing the slides 70 (in the slideholder 1) tissue side
down on top of the well 24 being careful that air bubbles are not
trapped and that contact is made between the solution in the well
24 and the tissue sample. Incubate overnight at 37.degree. C.
Inactivate the RNase-free DNAse with a 1 minute wash in DEPC water
and a 1 minute wash in 100% ethanol. Let the slides 70 air dry.
[0069] The reverse transcription is performed using the EZ RT PCR
system (Perkin Elmer). The RT/amplifying (RT-PCR) solution contains
EZ rTth buffer, 200 .mu.M each of dATP, dCTP, dGTP and dTTP, 400
.mu.g/mL bovine serum albumin, 40 Units RNasin, 0.8 .mu.M of 5' and
3' primers, 2.5 mM manganese chloride, 5 Units of rTth, and
2.times. concentrated Self-Seal Reagent (MJ Research, Inc.). Twenty
to fifty .mu.L of the RT-PCR mixture is placed into each of three
wells 24 in a specially designed in situ PCR aluminum tray 14 (the
depth of the wells 24 is approximately 0.005-0.03 mm) to fill the
wells 24. The slides 70 are carefully placed onto the wells 24 with
the tissue being placed in contact with the solution inside of the
well 24. The slides 70, slideholder 1 and aluminum tray 14 are
placed onto a block of a thermal cycler at 65.degree. C. for 30
minutes followed by a denaturation step at 94.degree. C. for 3
minutes. Twenty to 30 cycles are performed, each cycle being
60.degree. C. for 2 minutes followed by 94.degree. C. for 1
minute.
[0070] Following the cycling steps, the slideholder 1, slides 70
and aluminum tray 14 are placed vertically into a staining dish
with 2.times.SSC at 37.degree. C. for 5 minutes. The slideholder 1
is separated from the aluminum tray 14 and washed with
0.5-1.times.SSC at 37-60.degree. C. for 10-30 minutes (depending
upon background). In situ hybridization is performed as described
in Example 2 using a biotinylated or digoxigenin labeled probe
chosen internal to the primers.
[0071] Those of skill in the art recognize that amplification
schemes other than PCR are now well known and widely used and can
be used in place of PCR. These include ligation amplification (or
ligase chain reaction, LCR) and amplification methods based on the
use of Q-beta replicase. Also useful are strand displacement
amplification (SDA), thermophilic SDA, and nucleic acid sequence
based amplification (3SR or NASBA). See, e.g., U.S. Pat. Nos.
4,683,195 and 4,683,202 and Innis et al. (1990) for PCR; Wu and
Wallace (1989) for LCR; U.S. Pat. Nos. 5,270,184 and 5,455,166 and
Walker et al. (1992) for SDA; Spargo et al. (1996) for thermophilic
SDA and U.S. Pat. No. 5,409,818, Fahy et al. (1991) and Compton
(1991) for 3SR and NASBA.
Example 4
Wells with Multilayered Dried Reagents
[0072] Assays can be performed with a single reagent predried in a
well 24 and if the use of several reagents is required, the slide
70 with biological sample can be moved from a first well 24 with
the first reagent to a second well 24 with the second reagent,
etc., wherein the various wells 24 can either be on the same or on
separate trays 14. Alternatively, more than one reagent may be
predried in a well 24. The reagents can be dried in layers with the
outermost layer being the first reagent to be used. This is
demonstrated in FIG. 4 which shows a slide 70 with cells or tissue
section 220 placed over a well 24 into which has been predried in
order: a secondary antibody 270, a primary antibody 260, and a
protein blocking reagent 250. In this manner, different reagents
are separated and dry stored thereby preventing reaction until the
addition of water or buffer to the well. Upon addition of water (if
salts are predried in the well) or buffer to the well, the protein
blocking agent 250 will dissolve first since it was in the final
layer of reagents predried in the well 24. Next the primary
antibody 260 will dissolve and finally the secondary antibody 270
will dissolve and be able to react. Such a system allows all three
steps to occur without the necessity of moving the slides 70 from
one tray 14 to another tray 14 or from one well 24 to another well
24. For a different type of assay, for example one which requires a
series of four reagents, one may either predry all four reagents in
reverse order of action in a single well 24 or it may be found that
the use of two trays each with two reagents or one tray with three
reagents and a second tray with either the first or fourth reagent
works better, for example when a wash step is needed between the
step or steps of the first tray and the step or steps of the second
tray. Other variations on these schemes are obvious to one of skill
in the art. Any such combination requires less manual labor then
the use of four separate trays. Especially in the field of
pathology for which the types of assays to be performed are well
standardized, such a system is quite amenable to mass production of
trays with predried reagents which can then be stored until time of
use. This system is not limited to the use of antigen/antibody
reactions but can also be used for other reactions, e.g., enzymes
can be dried in the wells, nucleic acid hybridization can be
performed with different probes dried in the wells, a fluorescent
probe can be the dried reagent, biotin can be dried in the well,
etc.
[0073] To prepare wells with multiple layers of different reagents,
it is preferred to include layers of inert material between the
layers of reagents. For example, a well may be coated with reagents
as follows. A secondary antibody is coated onto a well and allowed
to dry. On top of this is coated a high concentration of an inert
material (i.e., a material not necessary for any of the reactions
and which will not interfere with the reactions) such as bovine
serum albumin, gelatin, sucrose, fetal calf serum, starch, agarose
or other inert material. This is allowed to dry. It is preferred
that the inert material be added in several layers, e.g., gelatin
in solution is added, allowed to dry, then more gelatin in solution
is added, allowed to dry, etc. This can be performed as often as
desired, the number of layers affecting the delay time until the
release of the secondary antibody. Five such coatings on top of the
secondary antibody has been found to give good results with a delay
of about 15-20 minutes until the release of the secondary antibody
from the time this inert layer begins to dissolve. On top of this
first layer (or multilayer) of inert material is coated a primary
antibody which is allowed to dry. On top of the primary antibody is
coated a second layer or multilayer of inert material. This can be
a low concentration of bovine serum albumin, gelatin, fetal calf
serum, starch, agarose or other inert material. Three coatings of
this second inert layer has been found to yield good results with a
delay time of about 10 minutes until the release of the primary
from the time the second inert layer begins to dissolve. On top of
the second inert layer is coated a protein block such as horse and
goat serum. The protein block is allowed to air dry. The
multilayers of inert material take time to dissolve thereby giving
each reaction enough time to occur prior to the next layer of
active reagent dissolving.
[0074] The limitation of this system is that it can only be used
for a series of steps which do not require a wash step in between
successive steps. For example, if reaction with a primary antibody
is followed by reaction with a secondary antibody, the secondary
antibody must be washed off prior to the detection step. Therefore
the detection reagent cannot be predried in the same well as the
secondary antibody. Similarly, if one step requires heating (e.g.,
denaturation of a nucleic acid probe) this cannot be combined with
a reagent which is heat inactivated or destroyed.
Example 5
Built-In Controls and Automatic Labels
Immunoassays or ISH/FISH
[0075] When assays are performed in a clinical setting, controls
are required by the Food and Drug Administration. Having built-in
controls on the very slides being assayed is an excellent manner in
which to test the controls. If the control is on a completely
different slide, the control is not as good because it cannot
indicate whether there was a problem such as reagent not contacting
the biological sample on either the control or the actual test
sample or missing a step of adding a reagent to either the control
or the test sample. Also, the reagents dropped onto the control
sample may accidentally be different from those dropped onto the
test sample by a human or by machine error, especially when several
tests are being performed simultaneously. When the control is on
the same slide as the test sample, such problems will be indicated
by controls, but if the control is a section of normal or
neoplastic tissue it is very labor intensive and time consuming to
prepare the control sample.
[0076] FIG. 3 illustrates a slide 70 onto which a tissue slice 220
has been fixed and also illustrates a separate region of slide 70
onto which has been affixed a stamp or sticker 230 (e.g., a piece
of nitrocellulose or other membrane or plastic or glass type matrix
glued onto the slide 70) with six distinct regions A-F, although
the use of a stamp or sticker is not essential, e.g., the controls
can be directly coated onto the slide 70. Each region of A-F has
been spotted with, e.g., a distinct antigenic substance or nucleic
acid, depending on the type of assay being performed, although
these substances can be applied directly to a region of the slide
70 in lieu of using a stamp or sticker 230. Six separate assays are
to be performed using a six well tray. Each well 24 will have a
reagent A'-F' which reacts, respectively, with A-F. Control A
should be positive only on the slide 70 placed onto well 24 with
reagent A' and should be negative for the remaining 5 wells.
Control B should be positive only on slide 70 placed onto the well
24 with reagent B' and should be negative for the other 5 wells,
etc. The stamps or stickers 230 with these external controls can be
premade commercially for mass sale or they can be custom made. It
is also useful if a stamp or sticker 230 for a common clinical
panel of assays is color coded or otherwise labeled so that a quick
glance is indicative of the assays being performed. This color code
or other labeling can also be matched to the color code or other
labeling of trays 14 to be used in conjunction with the stamp,
e.g., a green stamp will have antigenic determinants A-F on it and
a green tray will have antibodies A'-F'. A numbering or lettering
system can be used as one alternative to a color coding scheme.
These could be used for a series of tests for breast cancer whereas
a red stamp and red tray could indicate those to be used to assay
for Hodgkin's disease. Any type of color coding, such as a series
of stripes of colors, can be used. Such color coding will result in
fewer errors being made in the clinical laboratory. The use of the
positive control on each slide also acts as an automatic labeling
system for the slide since the positive external control is
indicative of the assay performed for that slide. If desired, the
stamps can be packaged with their corresponding trays and can even
be placed onto each tray when packaged and then peeled from the
tray and placed onto a slide at the time of use. The use of such
stamps or stickers with controls on them is much simpler and less
time consuming than preparing a control biological sample, e.g., a
tissue section of normal or neoplastic tissue, to be used as such a
control.
[0077] As an example, a breast panel of assays can be performed in
which six distinct diagnostic markers are used. These diagnostic
markers can be cytokeratin 7, cytokeratin 20, ER, Bcl-2, PR, and
cathepsin D. Each of these antigenic determinants can be coated
onto a stamp or sticker to be used as controls and the
corresponding antibodies can be predried on separate wells of a 6
well tray. If cytokeratin 7 or an equivalent antigenic determinant
is placed on position A of the stamp or sticker, then antibody
against cytokeratin 7 is to be placed in well A'. Section A of the
stamp or sticker should be positive on the slide placed on well A'
but should be negative on the other 5 wells. Also, only section A
of the stamp should be positive on the slide 70 placed on well A',
while sections B-F of the stamp or sticker should be negative. This
results in the automatic labeling of the slide by the built-in
control. If section A is not positive or if any of sections B-F are
positive on this slide it means that a problem has occurred and the
test should not be relied upon.
[0078] Other examples of panels which may be used are a panel of
prognostic markers for breast cancer such as Ki-67, Her-2/neu
(c-erbB-2), P53, pS.sub.2, EGFR, and Factor VIII. Other neoplasms,
e.g., prostate, bladder and colon can also use the same prognostic
panel tray. In general pathology practice, four panel trays can
cover 90-95% of diagnoses of all hemopoietic diseases: 1) A
Hodgkin's disease panel may include the markers LCA (CD45), L26
(CD20), CD3, Leu-M1 (CD15), Ki-1 (CD30), and LMP. 2) A
non-Hodgkin's panel can include L26 (CD20), CD3, MT1, Bcl-1, Bcl-2,
Ki-1 (CD30). 3) A separate non-Hodgkin' s panel can include Kappa,
Lambda, UCHL-1 (CD45RO), CD5, CD23, and CD10. 4) A leukemia panel
can include L26 (CD20), CD34, MPO, Lyso, TdT, and DBA44. Any other
desired panel of tests can be similarly performed, such as but not
limited to, panels for undifferentiated tumor of unknown primary
site, sarcoma classification, lymphoma vs. carcinoma vs. melanoma,
adenocarcinoma vs. mesothelioma, hepatocellular/cholangiocarcinoma
vs. metastatic carcinoma, pituitary panel, Paget's disease vs.
melanoma vs. squamous cell carcinoma vs. fibrous histiocytoma,
breast panel, and bladder vs. prostate carcinoma. Yet other
possible panels are a neuroendocrine panel, small round cell tumor,
germ cell tumor, Hodgkin's vs. non-Hodgkin's lymphoma, lymphoma vs.
reactive hyperplasia, plasma cell dyscrasia, leukemia panel and a
virus panel.
[0079] Each laboratory can devise its own system which is most
appropriate to the personnel and to the number and types of assays
being performed. For example, if an assay requires use of a first
set of antibodies followed by reaction with a secondary antibody
wherein the secondary antibody is identical for all samples, then
if a small number of assays are to be performed one may do these on
the trays 14, but if a large number of assays are being performed
one may prefer to place all the slides into a large tank with the
secondary antibody and/or detection system (a "batch" or "bulk"
incubation method. Alternatively, for the lab doing a small number
of assays, it is possible to coat a piece of filter paper with the
secondary antibody and/or detection system, lay all the slides onto
the filter papers and wet the filter paper at the time of use. This
can be less expensive than using the trays. Similarly, nucleic acid
probes can be placed onto the filter paper.
Example 6
Built-In Controls
Nucleic Acid Hybridization
[0080] In a manner similar to that discussed in Example 5 for
immunoassays, built-in controls can be used for nucleic acid assays
such as ISH or fluorescent in situ hybridization (FISH). In one
type of FISH, fluorescent probes are used which illuminate large
portions of the chromosomes. This is referred to as whole
chromosome painting (WCP). This technique is useful for observing
gross chromosomal aberrations such as translocations. The probes
used can be in conjunction with a variety of different colored
fluorophores. For example, probes to chromosome 1 can fluoresce
orange, probes to chromosome 2 can be made to fluoresce green and
probes to chromosome 3 can use a red fluorescing fluorophore. It is
therefore possible to stain for all three chromosomes
simultaneously and still be able to easily distinguish them from
each other. In human cells, there can be up to 24 distinct nuclear
chromosomes, these being chromosomes 1-22, X and Y. If three
different fluorophores are used, all 24 chromosomes can be studied
by using only 8 different tissue sections or 8 different sets of
cells. These can be studied on 8 separate slides or if desired
several tissue sections or sets of cells can be placed on separate
sections of a single slide. It is possible to place 8 tissue
sections on a single slide and thereby study all 24 chromosomes on
a single slide with all reactions being performed simultaneously
using 8 different sets of three mixed probes. These can be tested
on a single cell smear slide by placing the slide on a tray or chip
with 8 separate wells wherein each well has had predried in it a
different set of 3 probes. Using microarray techniques, 24 built-in
controls will be directly coated on the slide such that they will
surround, within the inner borders, each well region (see FIG. 6E).
One of skill in the art recognizes that it is not necessary to use
8 sets of 3 probes. Other variations are possible such as 6 sets of
4 differently labeled probes. It is also not necessary to use trays
with predried reagents, rather the reagents can be added to the
trays in liquid form. In a similar fashion, other techniques, such
as in situ hybridization, can be performed using a desired number
of controls which have been directly coated onto the slide in the
region surrounding the inner borders of the wells. Although the
controls have been shown as placed on the slide so as to surround
the edges of the wells, such a pattern is not required and other
patterns of arranging the controls can be used so long as they are
in a region which contacts the reagents in the wells.
Example 7
Automated Multiwell Tray and Machine
[0081] Analysis of biological samples is very labor intensive, even
with the use of automated systems since the automated systems still
require several steps to be performed manually. A multiwell tray,
or a multiwell tray with predried reagents, attached to tubing and
a pump or pumps or connected to an automated processing machine can
be used to partially or completely automate the processing of
biological samples. Such a multiwell tray can be similar in design
to the tray 14 discussed earlier. But the automated multiwell tray
330 (see FIGS. 5A-B) is used for steps such as washing or with less
expensive reagents which can be used in larger amounts. The
reaction chamber 280 of the automated multiwell tray 330 is
designed to hold volumes such as 0.01-1 mL, although this amount is
not critical and can be larger or smaller. The well includes one or
more inlets and one or more outlets to accommodate tubing. The
tubing entering an inlet is attached to a pump. A slideholder 1
with attached slides 70 is placed on top of the automated multiwell
tray 330 and fluids can be pumped into the reaction chambers 280
through an inlet such as 300 or 302. Reagents can be recirculated
during the reaction time and reused if desired (e.g., as shown in
FIG. 5B) by using a pump 290 and tubing 295 through inlet 302 in
conjunction with tubing 310 through outlet 294. Alternatively one
can send the used material directly to a waste container 291 or a
sink or to be analyzed, such as on a gel or by other
instrumentation, via outlet 296. Circulated reagents can reduce
incubation or reaction time and reduce background. The
concentration of circulated reagents also can be gradually
increased or decreased to reach the optimal reactive condition,
especially when using multiple probes. This is especially
applicable when a soft bottom tray is used which allows the use of
varied volumes.
[0082] A central processing unit 286 controls the pumping of
reagents and can open and close valves on various pieces of tubing
attached to a pump so that one pump can control several different
reagents or alternatively multiple pumps can be used all controlled
by the central processing unit. With this setup, a slideholder with
slides and mounted biological samples can be placed onto a
multiwell tray, the central processing unit can be activated to
pump desired fluids and reagents into the reaction chambers either
recirculating the fluids or disposing of the fluids directly.
Different reagents can be pumped into the reaction chamber
sequentially without the need of a person transferring the slides
from one tray to another tray. For example, slides with biological
samples can be placed onto the automated multiwell tray and the
system can pump in the reagents: xylene, 100% ethanol, 90% ethanol,
hydrogen peroxide, a secondary antibody, detection reagents (ABC),
diaminobenzidine, hematoxylin, PBS wash solution between each step,
and the further 90% ethanol, 100% ethanol and xylene and a
coverslipping solution. The slides can be removed from the
automated multiwell tray for any desired intervening steps for
which it is desirable to have the reaction performed on a regular
multiwell tray 14 as described earlier.
[0083] As another example, slides with a mounted tissue section can
be deparaffinized and treated separately and then placed onto a
multiwell tray which has predried reagents and then be attached to
the automatic processing machine which will pump in the desired
reagents, e.g., secondary antibody, detection reagents (ABC),
diaminobenzidine and hematoxylin as well as PBS wash buffer between
each of these steps, followed by 90% ethanol, 100% ethanol, xylene
and a coverslip solution.
[0084] The use of the automated multiwell tray has several
advantages. It allows several steps to be done in succession with
no manual labor required at each step. It also is safer because
some dangerous chemicals, e.g., xylene and diaminobenzidine which
are carcinogens, can be pumped directly from a container into the
reaction chamber and from there into a waste receptacle or a
receptacle from which the reagents can be reused without the need
of a person pipetting these reagents into wells and handling the
trays with these carcinogens on them. Recycling of such reagents
using the prior art method of simply dropping reagents on top of
biological samples mounted on slides is impracticable. Therefore
the automated multiwell tray reduces exposure to hazardous
chemicals, makes it easy to dispose of hazardous chemicals, and
also reduces use of such chemicals because they can be reused and
recycled.
[0085] The central processing unit 286 can also control heating and
cooling of a heat block 288 to perform automated in situ PCR or to
denature a probe being used for in situ hybridization. PCR
reagents, including biotin or digoxigenin if desired, and primer
sets can be coated and dried onto the wells of the tray 330. The
slide 70 with sample 220 is placed onto the tray 330 and water or
buffer is added. The heating block 288 can be placed against the
slide 70 (as shown in FIG. 5B) or the tray 330 or can be one
designed to contact both sides of the slide plus tray assembly and
can be controlled by the central processing unit 286. Two results
can be obtained from each well 410. First, fluid from a well 410
can be removed and assayed on a gel 298 to determine whether a band
of DNA is seen. The size of any such band can also be determined on
the gel 298. This acts as a control to see whether the PCR has
worked successfully. This is possible because a large fraction of
the amplified DNA does not remain in the cells of the sample but
leaks out to the fluid in the well. Second, a fraction of the
amplified DNA remains in the cells and this can be observed by
detecting the biotin or digoxigenin by methods well known to those
of skill in the art. Thus an in situ PCR shows which cells are
detected by the assay.
[0086] The present invention also uses a novel modification which
allows one to recover the reaction fluid and to assay this fluid,
prior to continuing the work-up of the tissue sample, to determine
whether the PCR has worked properly or has been contaminated. This
assay is extremely quick and simple, e.g., simply running the
reaction fluid on an agarose gel and looking for the presence of a
specific band size. In the event that one determines that the PCR
did work properly, then it is worth continuing the workup of the
tissue sample. However, if it is determined that the PCR failed,
one knows that it is not worth the labor and expense of continuing
with the particular sample.
[0087] The above noted ability to assay the reaction fluid is
useful not only for determining whether it is worth continuing to
workup the specific sample, but this ability also yields data not
available from viewing only the in situ hybridization results
within the tissue. When in situ hybridization is performed, some
fraction of amplicons remains where it was amplified while the rest
ends up in the solution. By assaying the portion in solution, one
can determine not only a relative amount of nucleic acid, but one
is also able to determine the size of the amplified nucleic acids.
When one views only the tissue sample one cannot determine the size
product which is formed, one learns only that some nucleic acid was
amplified and one also learns which cells were expressing the
nucleic acid. These two sets of data are complementary. It is
apparent that the present invention allows one to view both sets of
results with the data of both being complementary. To date no
apparatus has been available which had allowed one to obtain both
types of data from a single polymerase chain reaction.
[0088] A further aspect of the invention is that the volume of the
reaction chamber 280 is adjustable.
[0089] Preferably a central processing unit 286 controls a piston
284 which pushes against reaction chamber bottom 282 which is
either flexible or movable. This movement adjusts the volume of
space in the reaction chamber 280. For example, when performing in
situ PCR, it is desirable to keep the reaction volume very small,
e.g., 10-50 .mu.L. Following the PCR reaction it may be desired to
pump the reaction fluid out of the reaction chamber. However, such
a small volume of fluid will be held between the slide 70 and
reaction chamber bottom 282 by capillary action. By allowing the
reaction chamber to be enlarged to encompass more fluid, it becomes
easier to accomplish the desired pumping. Those of skill in the art
recognize that a variety of means can be used to adjust the volume
of the reaction chamber 280. It is not necessary to use a piston
controlled by a central processing unit. For example a screw means
can be placed against the reaction chamber bottom and by turning
the screw means the screw means will press against the tray bottom
to force the bottom of the reaction chamber toward the microscope
slide to reduce the volume of the reaction chamber 280. Reversal of
this process again enlarges the volume.
Example 8
Whole Chromosome Painting
[0090] Chromosomes can be examined for gross abnormalities such as
translocations by a technique known as whole chromosome painting.
This method uses a number of fluorescently labeled probes which
bind to a chromosome effectively to "light up" the whole
chromosome. Sets of probes specific for each chromosome can be used
to study any desired chromosome. Humans have a total of 24 nuclear
chromosomes, these being chromosomes 1-22, X and Y. It is common to
paint multiple chromosomes at one time. The chromosomes are easily
distinguished by using fluorescent probes of different colors. For
example, chromosomes 1, 2 and 3 can be stained simultaneously by
using probes which fluoresce orange for one chromosome, probes
which fluoresce green for a second chromosome, and probes which
fluoresce red for a third chromosome. Using such a system, one test
would typically use 8 slides of cells to examine the complete
nuclear genome of a human. This test would include the placing the
8 slides onto 8 wells of a tray. One example of tissue to be
assayed is a blood or bone marrow smear. The probes can be predried
in the wells if desired.
[0091] A chip or tray 400 designed to allow the analysis of all 24
chromosomes on a single slide 70 is presented here. The tray 400 is
one which can snap on to or otherwise be attached to a microscope
slide 70. The chip or tray 400 contains 8 wells 410 with each well
410 separated from neighboring wells 410 by a gap or a trough 420.
Such a tray 400 is illustrated in FIG. 6A. Each well 410 in the
tray 400 has a narrow opening 430 through which reagents can be
added to the wells 410.
[0092] In practice, cells to be examined are dropped or spread
across a microscope slide 70. The slide 70 is then attached to the
tray 400 such that the cells are facing the wells 410 of the tray
400. Reagents are then added to each well 410 individually through
the opening 430 in the tray to each well 410. The reagents will
spread between the well 410 and the slide 70 by capillary action.
Different reagents specific for the various chromosomes are added
to each well 410. The gap or trough 420 between wells 410 prevents
the reagents from one well 410 spreading to a neighboring well 410
thereby preventing cross-contamination. The wells 410 hold a
predetermined amount of fluid, e.g., 10-20 .mu.L each, and
capillary action allows only enough buffer to be added to fill the
wells 410 without causing excess overflow. This aids in preventing
cross-contamination. Three different chromosomes can be assayed in
each well 410 using, e.g., orange, green and red fluorescent probes
thereby allowing all 24 human nuclear chromosomes to be assayed on
a single slide 70.
[0093] In a preferred embodiment, the probes are predried onto the
8 wells 410 of the tray 400 with probes for 3 different chromosomes
in each well 410. If desired, other reagents such as salts can also
be predried into each well 410. Metaphase or interphase cells are
fixed across a slide 70 and the slide 70 is placed in contact with
the tray 400. Then buffer is added to the openings 430 to each well
410. With this method, there is no necessity to pipet the different
reagents into each well 410, rather the same buffer is added to all
wells 410 thereby preventing the possibility of pipetting incorrect
reagents (human error) into wells 410. The predried probes and
salts dissolve upon addition of buffer to the wells 410 and
hybridization is allowed to occur. A typical incubation may be at
70-90.degree. C. for 1-2 minutes to denature the probes as well as
the cellular DNA followed by an incubation at 37-45.degree. C. for
approximately 2 hours, although it is common to perform incubations
for anywhere from 30 minutes to overnight. The hybridization buffer
can be chosen as desired with several buffer systems commonly used
in the art. For example 2.times.SSC is commonly used. Formamide is
sometimes added to the buffer. In a preferred embodiment, following
incubation the tray 400 can be placed onto a blotting material,
e.g., paper towels, and the reaction fluid in the wells 410 will be
physically removed from the wells 410 by capillary action, the
blotting material soaking up the hybridization fluid. This prevents
cross-contamination between wells 410 when the slide 70 is
separated from the tray 400.
[0094] In a more preferred embodiment, the slide 70 includes
positive and negative controls in the regions 440 which are those
which are in contact with the hybridization fluid in each of the 8
wells 410. Using microarray technology which has become quite
popular recently, nucleic acids which are complementary to the
probes being used to paint the chromosomes are coated and
immobilized onto the slide 70, preferably prior to placing cells
upon the slides 70. This may best be performed under industrial
conditions and the slides 70 can be sold with the controls built
in. It is preferred that 24 controls 442 are placed onto each slide
70 at all 8 regions which are to be in contact with hybridization
buffer. One example of an array is shown in FIG. 6E in which all 24
nucleic acids are arrayed around the edges of each region 440 which
will contact each of the 8 wells 410. If for example, a first
region 440 is one which will contact a well 410 containing probes
for chromosomes 1, 2 and 3, then the control nucleic acids for
these chromosomes should light up after staining (each showing only
a single color) while the remaining 21 controls should not
hybridize and should not fluoresce. In this manner there are both
positive and negative controls and labels for each of the 8 wells
410.
[0095] One of skill in the art recognizes that other similarly
designed trays can be utilized. There is no need for an 8 well
tray. For example, if 4 differently colored fluorescent probes are
to be used, the same results could be obtained with a 6 well tray.
Furthermore, this invention is not limited to the analysis of human
chromosomes. Chromosomes from any other organism can be similarly
examined and the number of wells on the tray is a matter of
personal choice, often determined by the number of chromosomes or
probes to be examined. One of skill in the art also recognizes that
trays can be designed to hold more than a single slide such that
multiple cell samples can be assayed at once, with the multiple
slides being handled together more easily than several separate
slides.
Example 9
Coverslip with Concave Wells
[0096] Rather than using a method of simply dropping reagents onto
biological samples mounted onto a slide or placing the slide onto a
tray with wells which are filled with reagents, a slide or series
of attached slides can be covered with a coverslip wherein the
coverslip is concave thereby comprising one or more wells. This is
illustrated in FIGS. 7A-E which illustrates samples on six slides
being analyzed simultaneously. FIG. 7A shows slides 510 with
mounted biological samples 520 held in slideholder 515. FIG. 7B
illustrates a coverslip 500 which is to fit over the slides 510 of
FIG. 7A. Insert 540 discussed below may include writing 501 which
can display information. Regions 502 and 503 are positive and
negative controls, respectively. Controls 502 and 503 can be, e.g.,
protein, nucleic acid or a cell line, depending upon the specific
type of assay being performed. Channels to allow the inlet of
liquids and the outlet of air are shown as 504 and 505. The well
530 is also illustrated. The coverslips 500 can also be labeled
with a barcode, shown in FIG. 7B as 506 or can have text written on
them.
[0097] FIG. 7C shows coverslip 500 placed onto slides 510. The
coverslip 500 is placed onto the slide 510 with mounted biological
sample 520 and is affixed to the slide 510 at the top portion of
the coverslip 500. The slide 510 and coverslip 500 are then dipped
into water, buffer or reagent. Capillary action will cause the
liquid to rise into the well 530 of the coverslip 500. Surface
tension will hold the coverslip 500 securely to the slide 510. This
results in an enclosed system with a known volume and concentration
of reagent.
[0098] FIG. 7D illustrates the results after reaction has occurred
and the coverslip 500 has been removed. The biological samples 520
and the positive controls 502 are shown as being stained.
[0099] In a preferred aspect of the invention, the coverslip 500
has had reagent or reagents predried onto it. When a coverslip 500
with predried reagent is placed onto a microscope slide 510 with
biological sample 520, the slide 510 and coverslip 500 are merely
dipped into water or buffer thereby causing liquid to fill the well
530 of the coverslip 500 and dissolve the dried reagent. The slide
510 and coverslip 500 are then removed from the water or buffer and
the reaction is allowed to proceed. Known amounts of reagent or
reagents are predried thereby resulting in precisely known amounts
of reagents within the well 530 and thereby in contact with the
biological sample 520. The volume of the well 530 is also known
thereby resulting in a known concentration of reagent.
[0100] In another preferred aspect of the invention, the coverslip
500 is attached to the slide 510 by gluing an insert 540, e.g.,
glass or plastic, to the slide 510 using a glue which is resistant
to both organic and aqueous liquids. This is illustrated in FIGS.
7E-H for a single slide and coverslip for a single slide. Coverslip
500 including well 530 with channels 504 and 505 is placed onto
insert 540. FIG. 7F illustrates insert 540 which includes positive
502 and negative 503 controls, writing 501 to identify the insert
540, and a region of water-soluble glue 542. The upper portion of
the coverslip 500 is thereby glued to the insert 540 using a glue
which is water soluble. Controls 502 and 503 are located such that
they are within the well 530 region of the coverslip 500. The back
side of insert 540 is placed against and affixed to slide 510 by
means such as a glue which is resistant to both organic and aqueous
solutions. The slide 510 plus coverslip 500 is dipped into buffer
and removed and the reaction is allowed to proceed. The slide 510
plus coverslip 500 can then be processed by placing into tanks of
reagents or wash solution. Aqueous solutions will cause the water
soluble glue to dissolve thereby releasing the coverslip 500 but
not the insert 540. The coverslip 500 is easily removed at this
point. Insert 540 remains on slide 510 as a control and label.
[0101] In a further aspect of the invention, the slides 510 have
control samples 502 and 503 affixed to them. The controls 502 and
503 can either be spotted onto the slides 510, be on pieces of
paper or stamps which are glued to the slide 510, or they can be on
the insert 540. These control samples, which can be positive
controls, negative controls, or both (affixed as separate spots)
are used to determine that the reactions have worked properly. If
the controls 502 and 503 are affixed to the insert 540, they are
affixed at a point which will not be covered by glue and which
overlaps the well 530 of the coverslip 500 so that the control
samples 502 and 503 are in contact with buffer and reagents.
[0102] The inserts 540 can be premade with controls 502 and 503 and
then used when needed. These inserts 540 can further include
writing to indicate the names of the controls 502 and 503 and
whether they are positive or negative.
[0103] The coverslips 500 can also be labeled and may include bar
codes 560 for easy or automated reading. Coverslips 500 with
predried reagents are easily stored and are ready for use making
their use very convenient. Use of coverslips 500 with predried
reagents further means that pipetting of small, accurate amounts of
reagents is not required at the time of analysis thereby allowing
faster analysis of the biological samples.
Example 10
Automated Method for Processing Biological Samples on Slides
[0104] A method similar to that of Example 9 can be automated such
as by using a reaction chamber as illustrated in FIGS. 5A-B. One
difference is that the coverslip to be used in the automated
procedure need not include a well but can be flat. FIGS. 8A-D
illustrate the method. Slides 600 with biological samples 610 are
placed into slideholder 620. Coverslip 630 includes region 640
which can contain written information. Control samples 650 and 660
can be included on the coverslip 630. The coverslip 630 can also
include a barcode 670 or can include text written on it. Slides 600
with biological samples 610 are placed into a reaction chamber,
e.g., as shown in FIGS. 5A-B, for processing with organic reagents
to deparaffinize the samples 610. In a preferred embodiment,
several slides 600 are placed into a single slideholder 620 as
shown in FIG. 8A. After deparaffinizing the samples 610 and
washing, reagents can be added to the reaction chamber. In a
preferred embodiment, coverslip 630 is placed into the reaction
chamber together with slides 600. This is illustrated in FIG. 8C
which shows both the coverslip 630 and slideholder 620 with slides
600, although the reaction chamber is not illustrated. Coverslip
630 preferably has reagent predried onto it, preferably in region
680. Addition of water or buffer dissolves the reagent which then
reacts with biological sample 610 as well as with control samples
650 and 660. Following reaction, wash solutions can be passed
through the reaction chamber. Upon completion of the wash, the
coverslip 630 can be pushed against slides 600 which are removed
together from the reaction chamber and are kept together, i.e., the
coverslip 630 acts as a permanent coverslip unlike the coverslip
500 in Example 9. FIG. 8D shows the coverslip 630 mounted onto
slides 600 with the biological samples 610 and positive controls
650 being positive.
[0105] The preferred method of predrying known amounts of reagent
onto the coverslip 630 allows for very quick and easy use in a
clinical laboratory. The reagents need not be measured or pipetted.
Instead a coverslip 630 is simply dropped into a reaction chamber
together with the slide 600 with biological sample 610 and the
reaction is allowed to proceed. Furthermore, the coverslip 630 can
include positive and negative controls prespotted on to it thereby
allowing for simple analysis of whether the reaction has worked
properly.
[0106] Use of the above methods allows one to obtain results of a
whole panel of markers in as little as 15-30 minutes. Thus the
results can be obtained while the patient is still in the operating
room. The pathologist and surgeon can decide immediately whether to
perform more surgery or if chemotherapy or radiation treatment is
necessary. This can allow the surgeon to proceed immediately rather
than having to perform more surgery at a later date. If the
currently sold automated system were used instead of the methods of
the instant invention, it would take longer to receive results,
partially because the currently sold automated system does not
assay one patient at a time but rather many samples are loaded into
the automated instrument at one time and it is necessary to wait
while they are all loaded and then processed. The currently sold
automated system drops reagents on top of slides and the biological
sample is not always completely covered, whereas the present method
of placing a biological sample on top of a well filled with
reagents ensures that the whole sample is in contact with
reagent.
[0107] The above Examples are only exemplary and not meant to be
limiting of the techniques which may be performed using the
apparatus which is defined by the present invention. The invention
is applicable to, but not limited to, immunohistochemistry, in situ
hybridization, in situ PCR, and fluorescent in situ hybridization
(FISH). The stated measurements are also exemplary and not meant to
be limiting as it will be obvious to one of skill in the art that
the exact measurements are not critical and can be varied to still
yield successful results. Those skilled in the art will readily
perceive other applications for the present invention.
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[0113] Spargo C A, et al. (1996). Mol. Cell. Probes 10:247-256.
[0114] Walker G T, et al. (1992). Nucl. Acids Res. 20:1691-1696.
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