Note: Descriptions are shown in the official language in which they were submitted.
~
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Removal of Embedding Media from Biological Samples and Cell
Conditioning on Automated Staining Instruments
10
BACKGROUND OF THE INVENTION
Field of the Invention
The present invention relates to a method for removing embedding media from
biological samples on automated instruments prior to immunohistochemical
(IHC), in situ
hybridization (ISH) or other lustochemical or cytochemical manipulations. The
present
invention also relates to a method for conditioning cells or tissues so as to
increase the
accessibility of various molecules to their respective targets and generally
to improve tissue
and cell readability.
Summary of the Related Art
Diagnosis of disease based on interpretation of tissue or cell samples taken
from a
diseased organism has expanded dramatically over the past few years. In
addition to
traditional histological staining techniques and immunohistochemical assays,
in situ
techniques such as in situ hybridization and in situ polymerase chain reaction
are now used
to help diagnose disease states in humans. Thus, there are a variety of
techniques that can
assess not only cell morphology, but also the presence of specific
macromolecules within
cells and tissues. Each of these techniques requires the preparation of sample
cells or
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tissues which may include fixing the sample with chemicals such as an aldehyde
(such as
formaldehyde, glutaraldehyde), formalin substitutes, alcohol (such as ethanol,
methanol,
isopropanol) or embedding the sample in inert materials such as paraffin,
celloidin, agars,
polymers, resins, cryogenic media or a variety of plastic embedding media
{such as epoxy
resins and acrylics). Other sample tissue or cell preparations require
physical manipulation
such as freezing (frozen tissue section) or aspiration through a fine needle
(fine needle
aspiration (FNA)). Regardless of the tissue or cell sample or its method of
preparation or
preservation, the goal of the technologist is to obtain accurate, readable and
reproducible
results that permit the accurate interpretation of the data. One way to
provide accurate,
readable and reproducible data is to prepare the tissue or cells in a fashion
that optimizes
the results of the test regardless of the technique employed. In the case of
immunohistochemistry and in situ techniques this means increasing the amount
of signal
obtained from the specific probe (antibody, DNA, RNA). In the case of
histochemical
staining it may mean increasing the intensity of the stain or increasing
staining contrast.
Without preservation, tissue samples rapidly deteriorate such that their use
in
diagnostics is compromised shortly after removal from their host. In 1893,
Ferdinand Blum
discovered that formaldehyde could be used to preserve or fix tissue so that
this tissue could
be used in histochemical procedures. The exact mechanisms by which
formaldehyde acts
in fixing tissues are not fully established, but they involve cross-linking of
reactive sites
within the same protein and between different proteins via methylene bridges
(Fox et al., J.
Histochem. Cytochem. 33: 845-853 (1985)). Recent evidence suggests that
calcium ions
also play a role (Morgan et al., J. Path. 174: 301-307 (1994)). These links
cause changes in
the quaternary and tertiary structures of proteins, but the primary and
secondary structures
appear to be preserved (Mason et al., J. Histochem. Cytochem. 39: 225-229
(1991)). The
extent to which the cross-linking reaction occurs depends on conditions such
as the
concentration of formalin, pH, temperature and length of fixation (Fox et al.,
J. Histochem.
Cytochem. 33: 845-853 (1985)). Some antigens, such as gastrin, somatostatin
and
a-1-antitrypsin, may be detected after formalin fixation, but for many
antigens, such as
intermediate filaments and leukocyte markers, immunodetection after formalin
treatment is
lost or markedly reduced (McNicol & Richmond, Histopathology 32: 97-103
(1998)). Loss
of antigen immunoreactivity is most noticeable at antigen epitopes that are
discontinuous,
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i. e. amino acid sequences where the formation of the epitope depends on the
confluence of
portions of the protein sequence that are not contiguous.
Antigen RetrievalTM is a term that describes the attempt to "undo" the
structural
changes that treatment of tissue with a cross-linking agent induces in the
antigens resident
within that tissue. Although there are several theories that attempt to
describe the
mechanism of Antigen RetrievalTM such as loosening or breaking of
crosslinkages formed
by formalin fixation, it is clear that modification of protein structure by
formalin is
reversible under conditions such as high-temperature heating. It is clear that
several factors
affect Antigen RetrievalTM: heating, pH, molarity and metal ions in solution
(Shi et al., J.
Histochem. Cytochem. 45: 327-343 (1997)).
Microwave heating appears to be the most important factor for retrieval of
antigens
masked by formalin fixation. Microwave heating (100°t5°C)
generally yields better results
in Antigen RetrievalTM immunohistochemistry (AR-1HC).
Different heating methods have been described for antigen retrieval in IHC
such as
autoclaving (Pons et al, Appl. Immunohistochem. 3: 265-267 (1995); Bankfalvi
et al., J.
Path. 174: 223-228 (1994)), pressure cooking (Miller & Estran, Appl.
Immunohistochem. 3:
190-193 (I995); Norton et al., J. Path. 173: 371-379 (1994)); water bath
(Kawai et al.,
Path. Int. 44: 759-764 (1994)), microwaving plus plastic pressure cooking
(L1.S. Patent No.;
Taylor et al. (1995); Pertschuk et al., J. Cell Biochem. 19(suppl.): 134-137
(1994)), and
steam heating (Pasha et al., Lab. Invest. 72: 167A (1995); Taylor et al., CAP
Today 9: 16-22
(1995)).
Although some antigens yield satisfactory results when rnicrowaving is
performed
in distilled water, many antigens require the use of buffers during the
heating process.
Some antigens have particular pH requirements such that adequate results will
only be
achieved in a narrow pH range. Presently, most Antigen RetrievalTM solutions
are used at a
pH of approximately 6-8, but there is some indication that slightly more basic
solutions
may provide marginally superior results (Shi, et al., J. Histochem. Cytochem.
45: 327-343
( 1997)).
Although the chemical components of the Antigen RetrievalTM solution,
including
metal ions, may play a role as a possible co-factor in the microwaving
procedure, thus far,
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no single chemical has been identified that is both essential and best for
Antigen
RetrievalTM.
Many solutions and methods are used routinely for staining enhancements. These
may include but are not limited to distilled water, EDTA, urea, Tris, glycine,
saline and
citrate buffer. Solutions containing a variety of detergents (ionic or non-
ionic surfactants)
may also facilitate staining enhancement under a wide range of temperatures
(from ambient
to in excess of 100°C).
In addition to cell surface molecules that may be present on the exterior
portion of
the cell, other molecules of interest in IHC, ISH and other histochemicai and
cytochemical
manipulations are located within the cell, often on the nuclear envelope. Some
of these
molecules undergo molecular transformation when exposed to a fixative
(coagulative or
precipitive) such as formalin. Thus with respect to these molecules it is
desirable to not only
overcome the effects of fixation but also to increase the permeability of the
cell in order to
facilitate the interaction of organic and inorganic compounds with the cell.
Other tissue samples may not have been subjected to cross-linking agents prior
to
testing, but improved results with respect to these tissues is also important.
There are a
variety of non-fonmalin methods for preserving and preparing cytological and
histological
samples. Examples of these methods include but are not limited to a)
hematology smears,
cytospinsTM, ThinPrepsTM, touch preps, cell lines, Ficoll separations for
lymphocytes and
huffy coats etc. are routinely preserved in a many ways which include but are
not limited to
air-drying, alcoholic fixation, spray fixatives and storage mediums such as
sucrose/glycerin
storage medium. b) tissues and cells (either fixed or unfixed) may be frozen
and
subsequently subjected to various stabilizing techniques such preservation,
fixation and
desiccation. c) tissues and cells may be stabilized in a number of non-cross-
linking
aldehyde fixatives, non- aldehyde containing fixatives, alcoholic fixatives,
oxidizing agents,
heavy metal fixatives, organic acids and transport media.
One way to improve testing results is to increase the signal obtained from a
given
sample. In a general sense, increased signal can be obtained by increasing the
accessibility
of a given molecule for its target. As in the case for antigens found within
the cell, targets
within the cell can be made more accessible by increasing the permeability of
the cell
thereby permitting a greater number of molecules entry into the cell, thereby
increasing the
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probability that the molecule will "find" its target. Such increased
permeability is
especially important for techniques such as ISH, in situ PCR, IHC,
histochemistry and
cytochemistry.
Tissues and cells are also embedded in a variety of inert media (paraffin,
celloidin,
OCTTM, agar, plastics or acrylics etc.) to help preserve them for future
analysis. Many of
these inert materials are hydrophobic and the reagents used for histological
and cytological
applications are predominantly hydrophilic, therefor the inert medium may need
to be
removed from the biological sample prior to testing. For example, paraffin
embedded
tissues sections are prepared for subsequent testing by removal of the
paraffin from the
tissue section by passing the slide through various organic solvents such as
toluene, xylene,
limonene or other suitable solvents. Traditional deparaffinization uses
organic solvents,
which generally requires that the process be performed in ventilated hoods.
Furthermore
use and disposal of these solvents increases the cost of analysis and exposure
risk
associated with each tissue sample tested.
1 S Presently, there is no available technique for removing inert media from
sample
tissue by directly heating the slide in an automated fashion. Neither is it
currently possible
to remove inert media from sample tissue while conditioning the sample tissue
or cell in a
one-step automated staining process.
The methods of the present invention permit a) automated removal of embedding
media without the use of organic solvents, thus exposing the cells for
staining and thereby
reducing time, cost and safety hazards b) automated cell conditioning without
automated
removal of embedding media from the sample cell or tissue. c) a mufti-step
automated
process that exposes the cells, performs cell conditioning and increases
permeability of the
cytological or histological specimens, thereby increasing sample readability
and improving
interpretation of test data. The methods of the present invention can be used
for improving
the stainability and readability of most histological and cytological samples
used in
conjunction with cytological and histological staining techniques.
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SUMMARY OF THE INVENTION
The present invention relates to an automated method for exposing biological
samples for use in histological or cytological testing procedures by removing
the i
embedding media without the use of organic solvents.
The present invention further relates to an automated method for cell
conditioning,
thus improving the accessibility of molecules in biological samples.
The present invention also relates to an automated method for the simultaneous
exposing and cell conditioning of biological samples for histochemical and
cytochemical
applications.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
One embodiment of the present invention relates to the exposing of biological
1 S samples by removal of the inert materials in which biological samples have
been embedded
for preservation and support. In a preferred embodiment of the present
invention, paraffin
or other inert materials are removed from biological samples by heating one
side of the
biological sample. This may be accomplished by contact heating of the
microscope slide
on which the embedded biological samples have been placed. Other inert
materials that can
be removed from embedded biological samples include but are not limited to
agars and
cryogenic media. This process of removal of inert embedding media or etching
of
embedding media is referred to herein as exposing.
In a preferred method of the present invention, the paraffin-embedded
biological
sample laying on the glass slide is first heated by a heating element. The
heating
element exposes heat on one side of the biological sample (such as the thermal
platforms disclosed in U.S. Patent No. 6,296,809 issued on October 2, 2001
within an
automated staining instrument (U.S. patent nos. 6,045,759 issued on April 4,
2000 and
6,405,609 issued on June 18, 2002) such that the sample slide is dried and the
paraffin is
melted. Typically, the biological sample is placed on a top surface of a slide
(such as a
glass slide). The slide
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is then placed on top of the thermal platform, so that the bottom surface of
the slide is in
contact with the thermal platform. The thermal platform, via conduction, heats
the
bottom portion of the slide. After the heating of the biological sample, the
inert material
may be removed from the slide by a fluid (as a gas or liquid). For example,
the inert
material may be rinsed with DI water and a surfactant.
In another method of the present invention, a paraffin embedded biological
sample is placed on a glass microscope slide and the microscope slide is
placed on a
heating element. A reagent is placed on the biological sample slide, the
biological
sample slide is then exposed to elevated temperatures that will permit the
melting of the
inert material, and after which the inert material may be removed from the
slide by a
fluid (as a gas or liquid).
In a preferred embodiment of the present invention, reagents are used in
conjunction with heating the embedded biological samples. Suitable reagents
may
include, but are not limited to, de-ionized water, citrate buffer (pH 6.0-
8.0), Tris-HC1
buffer (pH b-10), phosphate buffer (pH 6.0-8.0), SSC buffer, APK WashTM,
acidic
buffers or solutions (pH 1-6.9), basic buffers or solutions (pH 7.1-14),
mineral oil,
Norpar, canola oil, and PAG oil. Each of these reagents may also contain ionic
or non-
ionic surfactants such as Triton X-100, Tween, Brij, saponin and sodium
dodecylsulfate.
In a method of the present invention, the temperature of the heating element
is
raised to a temperature in excess of the melting point of the inert material.
For example,
the melting point of pure paraffin is listed as 50-57° C in the Merck
index. Thus, in the
method of the present invention, the temperature is in excess of the melting
point of the
paraffin in which the biological sample is embedded. In a preferred method of
the
present invention, the temperature is raised in excess of 50° C to
about 130° C.
In a method of the present invention, the duration of time required to melt
the
inert material will vary according to the temperature used and the embedding
material.
Typically, in an automated system, a processor, such as a microprocessor, is
used in
conjunction with a memory. The amount of time and the temperature required to
melt
the paraffin is contained within a table contained in the memory.
The paraffin embedded biological sample is subjected to elevated temperatures
ranging from 5 minutes to 60 minutes. The heating element used in the method
of the
*-trademark
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present invention requires that sufficient contact be maintained between the
surface on
which the biological sample is placed and the heating element.
Another embodiment of the present invention relates to the exposing of
biological samples without removal of the inert materials in which biological
samples
have been embedded for preservation and support. In a preferred embodiment of
the
present invention, biological samples are readied for testing by contact
heating of the
microscope slide on which the embedded biological samples have been placed.
Other
inert materials that are not removed from embedded biological samples include
but are
not limited to plastic or celloidin embedding media and/or other polymers and
resins.
In a preferred method of the present invention, the embedded biological sample
laying on the glass slide is first heated by the heating element. The heating
element
exposes heat on one side of the biological sample, such as by using the
thermal
platforms disclosed in U.S. patent no. 6,296,809 issued on October 2, 2001
within an
automated staining instrument (U.S. patent nos. 6,045,759 issued on April 4,
2000 and
6,405,609 issued on June 18, 2002) such that the sample slide is dried.
In another method of the present invention, an embedded biological sample is
placed on a glass microscope slide and the microscope slide is heated on one
side (e.g.,
by placing the slide on a thermal platform). A reagent is then placed on the
biological
sample slide and the biological sample slide, with the reagent, is then heated
to a
specified temperature (ranging from ambient to greater than 100°C) and
for a specified
amount of time (ranging from 2 minutes to 12 hours). This will cause etching
of the
surface of the inert embedding material, and after which the etching reagent.
may be
removed from the slide by a fluid (as a gas or liquid). As discussed
previously, the
amount of time and the specified temperature may be stored in memory.
In the preferred method of the present invention, reagents are used in
conjunction with or without heating the embedded biological samples. Suitable
reagents
may include, but are not limited to, de-ionized water, citrate buffer (pH 6.0-
8.0), Tris-
HC1 buffer (pH 6-10), phosphate buffer (pH 6.0-8.0), SSC buffer, APK WashTM,
acidic
buffers or solutions (pH 1-6.9), basic buffers or solutions (pH7.1-14) mineral
oil,
Noipar, canola oil, and PAG oil. Each of these reagents may also contain ionic
or non-
ionic surfactants such as Triton X-100, Tween, Brij, saponin and sodium
dodecylsulfate.
*-trademark
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In the method of the present invention, the temperature of the heating element
is
set to an appropriate level for the drying or the etching of the embedded
biological
sample. For example, etching may be carried out with a basic solution of
methanol
sodium hydroxide (sodium methoxide) at temperatures ranging from ambient to
37°C.
In the method of the present invention, the duration of time required to etch
the
inert material will vary according to the temperature used and the embedding
material
(plastic or celloidin embedding media and/or other polymers and resins etc.).
In a
preferred method of the present invention the embedded biological sample is
subjected
to appropriate temperatures ranging from 2 minutes to 12 hours. The heating
element
used in the method of the present invention requires that sufficient contact
be
maintained between the surface on which the biological sample is placed and
the heating
element.
A preferred embodiment present invention also comprises an automated method
of cell conditioni~tg, either concurrent with, subsequent to or independent of
removal or
1 S etching of the inert embedding material from the biological sample.
Heating the
biological sample in an appropriate (organic or inorganic) reagent has been
found to
improve the accessibility of the reagent to the target molecule in the cell
(protein,
nucleic acid, carbohydrate, lipid, pigment or other small molecule). This
process of
improving accessibility of the reagent (organic or inorganic) to the molecular
target is
referred to herein as cell conditiosting.
In one method of the present invention, cell conditioning is accomplished
while
the biological sample is being exposed as described above. In this method of
the present
invention, a biological sample is placed on a glass microscope slide and the
microscope
slide is heated on one side (e.g., by placing the slide on a thermal platform)
within an
automated staining instrument (U.S. Patent Number 6,045,759 issued on April 4,
2000). A
reagent is placed on the biological sample and the temperature of the heating
element
may or may not be increased. The biological sample is exposed to the
appropriate
temperature for an appropriate duration of time that will permit the melting
or etching of
the inert material and permit cell conditioning of the biological sample to be
subsequently stained using histological or cytological techniques.
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The reagents used for cell conditioning can be the same as those for ~rposing
the embedded biological sample. For example, for DNA targets, a cell
conditioning
solution may be a solution of EDTA; a common temperature setting may be
95°C for a
duration ranging from 6-60 minutes. For protein targets, a cell conditioning
solution
may be a solution of boric acid buffer; a common temperature setting may be in
excess
of 100°C for a duration ranging from 6-60 minutes. For RNA targets, a
cell
conditioning solution may be a solution of SSC; a common temperature setting
may be
75°C for a duration ranging from 6-60 minutes. For histochemical
reactions, such as a
Hematoxylin and Eosin (H&E) stain, a cell conditioning solution may be treated
de-
ionized water; a common temperature may range from 60-80°C for a
duration of 6-30
minutes. A partial list of possible reagents appears in Analytical Morphology,
Gu, ed.,
Eaton Publishing Co. (1997) at pp. 1-40. The solutions should generally be of
known
molarity, pH, and composition. Sodium dodecyl sulfate (SDS), ethylene glycol
is
preferably added to the conditioning solution. In addition, metal ions or
other materials
may be added to these reagents to increase effectiveness of the cell
conditioning.
In another method of the present invention, cell conditioning is accomplished
subseguent to the biological sample being exposed as described above. In this
method
of the present invention a biological sample is placed on a glass microscope
slide and
the microscope slide is heated on one side (e.g., by placing the slide on a
thermal
platform) within an automated staining instrument (LT.S. Patent Number
6,045,759 issued
on April 4, 2000). ~ t~s method, the embedded biological sample laying on the
glass
slide is first heated by the heating element within an automated staining
instrument such
that the sample slide is dried and the embedding material is melted or etched
and
removed by the application of a fluid. Subsequent to exposing the biological
sample, an
appropriate reagent is applied in order to permit cell conditioning of the
biological
sample to be subsequently stained using histological or cytological
techniques.
The reagents used for cell conditioning can be the same as those for exposing
the embedded biological sample. For example, for DNA targets, a cell
conditioning
solution may be a solution of SSC; a common temperature setting may be
95°C for a
duration ranging from 6-60 minutes. For protein targets, a cell conditioning
solution
may be a solution of Phosphate buffer; a common temperature setting may be in
excess
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of 100°C for a duration ranging from 6-60 minutes. For RNA targets, a
cell
conditioning solution may be a solution of SSC; a common temperature setting
may be
75°C for a duration ranging from 6-60 minutes. For histochemical
reactions, such as a
Trichrome stain, a cell conditioning solution may be Bouins; a common
temperature
may range from 60-80°C for a duration of 6-30 minutes.
In yet another method of the present invention, cell conditioni~:g is
accomplished without the biological sample being exposed. In this method of
the
present invention, a biological sample is placed on a glass microscope slide
and the
microscope slide placed on a heating element within an automated staining
instrument.
A reagent is placed on the biological sample and the temperature of the
heating element
may or may not be increased. Cell conditioning of the biological sample may be
performed prior to being stained using histological or cytological techniques.
The reagents used for cell conditioning can be the same as those for exposing
the embedded biological sample. For example, for DNA targets, a cell
conditioning
solution may be a solution of Sodium Citrate; a common temperature setting may
be
90°C for a duration ranging from 6-60 minutes. For protein targets, a
cell conditioning
solution may be a solution of urea; a common temperature setting may be in
excess of
100°C for a duration ranging from 6-60 minutes. For whole cells, a cell
conditioning
solution may be a solution of methanol; a common temperature setting may be
ambient
for a duration ranging from 4-10 minutes. For histochemical reactions, such as
an Acid
Fast Bacilli (AFB) stain, a cell conditioning solution may be peanut oil; a
common
temperature may range from 60-70°C for a duration of 30-60 minutes.
The present invention also comprises cell conditioning of cytological preps,
such as fine needle aspirations (FNA) smears, touch preps, Ficoll, Cytospins~,
Thins
Preps~, cervical-vaginal pap smears, blood or body fluid films, etc., that are
neither
fixed with an aldehyde nor embedded in a matrix, such as paraffin. Many are
fixed in
an alcohol, such as methanol or ethanol, others will be sprayed with hair
spray or other
aerosol fixative and dried, and still others will be placed in cytological
fixatives, which
may include carbowax and Saccomanno's (organic or inorganic) reagent among
others.
The cells are either centrifuged or filtered to a slide or directly touched to
a glass slide
and smeared in some cases (PAP's) or applied directly against the slide (touch
preps).
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The term "Biological sample" is meant any collection of cells (either loose or
in
tissue) that can he mounted on a standard glass microscope slide including,
without
limitation, sections of organs, tumors sections, bodily fluids, smears, frozen
sections,
blood, cytology preps, microorganisms and cell lines.
The term "Stain" is meant any biological or chemical entity which, when
applied
to targeted molecules in biological sample, renders the molecules detectable
under
microscopic examination. Stains include without limitation detectable nucleic
acid
probes, antibodies, and other reagents which in combination or by themselves
result in a
colored end product (by bright field or fluorescence).
The following examples are presented for illustrative purposes only and are
not
intended, nor should they be construed, as limiting the invention in any way.
Those
skilled in the art will recognize that variations on the following can be made
without
exceeding the spirit or scope of the invention.
EXAMPLES
Example 1
Automated "Exposing" and "Cell Conditioning"
with Biological Samples Stained with H&E
Biological samples, including breast CH'TN33, stomach 1496, brain, tonsil and
kidney, that had been embedded in paraffin were exposed according to the
following
procedure: slides containing the above referenced biological sample were
placed on an
automated instrument (Ventana Medical Systems, Inc., Tucson, AZ) and subjected
to
the exposing protocol described below. Generally, the slides containing paraff
n
embedded biological samples were dry heated to 65° C for six (6)
minutes then rinsed
with lx citrate buffer, de-ionized water, lOmM phosphate buffer (pH = 6.3), or
lOmM
Tris-HCl buffer (pH = 7.4) each containing 0.1% Triton X-100.
Exposing Protocol 1
1. Incubate for 2 minutes
2. Rinse slide
3. Adjust slide volume and apply liquid coverslipTM
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4. Incubate for 6 minutes
5. Rinse slide
6. Adjust slide volume and apply liquid
coverslipTM
7. Increase temperature to 65.0 C
S 8. Rinse slide
9. Adjust slide volume and apply liquid
coverslipTM
10. Incubate for 4 minutes
11. Adjust slide volume and apply liquid
coverslipTM
12. Incubate for 4 minutes
13. Adjust slide volume and apply liquid
coverslipTM
14. - Incubate for 4 minutes
15. Rinse slide
16. Decrease temperature to 42.0 C
17. Adjust slide volume and apply liquid
coverslipTM
18. Incubate for 4 minutes
19. Rinse slide
20. Decrease temperature to 42.0 C
21. Adjust slide volume and apply liquid
coverslipTM
22. Incubate for 4 minutes
23. Rinse slide
After automated exposing, the biological sample was stained with hematoxylin
and eosin by the following method. Slides were placed in hematoxylin 1
(Richard Allen
Scientific, Kalamazoo, MI} for 1.5 minutes and then rinsed with running de-
ionized
water for one minute. Slides were then placed in acid alcohol clarifies
(Richard Allen
Scientific) for one minute and then rinsed with running de-ionized water for
one minute.
Slides were then placed in diluting an~unonia-bluing reagent for one minute
(Richard
Allen Scientific, Kalamazoo, MI) and then rinsed in running de-ionized water
for one
minute. Slides were then rinsed in 95% ethanol, and then placed in 2.5% eosin
Y
(Richard Allen Scientific, Kalamazoo, MI) for 2.5 minutes. The biological
samples on
the slides were dehydrated by exposing the biological sample to a 100% ethanol
bath for
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one minute. This process was repeated three times followed by exposure of the
biological sample to a xylene bath for three minutes, twice. After the
dehydration step
the biological sample was covered with a coverslip.
Control biological samples were deparaffinized by a traditional solvent-based
S deparaffinization technique. Biological sample place on microscope slides
and
preserved in paraffin were completely submersed in a xylene bath for five
minutes.
Slides containing biological sample were placed in a second xylene bath for
five
minutes. After removal from the second xylene bath, the slides were placed in
a 100%
ethanol bath for three minutes. Slides were then placed in a second 100%
ethanol bath
for three minutes and then placed in a 90% ethanol solution for two minutes.
The slides
were then placed in 80% ethanol for one minute followed by complete immersion
in
distilled water for one to three minutes. After deparaffinization, the
biological samples
were stained with hematoxylin and eosin as described above.
The biological samples that were deparaffinized by the solvent technique and
by
the automated heating technique were compared after staining by hematoxylin
and
eosin. Morphology on all sets of slide was acceptable and essentially
equivalent. The
tonsil and brain biological samples that were exposed by the automated heating
method
showed more intensified hematoxylin staining than the biological samples
deparaffinized by standard solvent techniques.
Example 2
Automated "Exposing" of Biological a Samples
with Simultaneous "Cell CoHditioning"
Biological samples of kidney Q-10 and tonsil T998D that had been formalin
fixed and embedded in paraffin were exposed according to the protocol
described in
Example 1. After automated exposing, the biological sample was subjected to
the DAB
paraffin protocol used for irnmunohistochemical staining. The protocol for DAB
staining is described below:
1. Incubate for 2 minutes
2. Rinse slide
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3. Adjust slide volume and apply liquid coverslipTM
4. Rinse slide
5. Adjust slide volume and apply liquid coverslipTM
6. Rinse slide
7. Adjust slide volume and apply liquid coverslipTM
8. Apply one drop of inhibitor
9. Incubate for 4 minutes
10. Adjust slide volume and apply liquid coverslipTM
11. Apply one drop of primary antibody
12. Incubate for 32 minutes
13. Adjust slide volume and apply liquid coverslipTM
14. Apply one drop of Biotinylated Ig
15. Incubate for 8 minutes
16. Rinse slide
17. Adjust slide volume and apply liquid coverslipTM
18. Apply one drop of Avidin-HRPO
19. Incubate for 8 minutes
20. Rinse slide
21. Adjust slide volume and apply liquid coverslipTM
22. Apply one drop of DAB and one drop DAB H202
23. Incubate for 8 minutes
24. Rinse slide
25. Adjust slide volume and apply liquid coverslipTM
26. Apply one drop of Copper
27. Incubate for 4 minutes
28. Rinse slide
The primary antibody used for the kidney Q-10 biological sample was Anti-
CD15 (Ventana Medical Systems, Inc. Tucson, AZ, Catalogue no. 250-2504). The
primary antibody used for the tonsil T998D biological sample was Anti-CD45R0
(Ventana Medical Systems, Inc. Tucson, AZ, Catalogue no. 250-2563). The DAB
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staining kit used was obtained from Ventana Medical Systems, Inc. Tucson, AZ,
Catalogue no. 250-001.
Control biological samples were deparaffinized by a traditional solvent-based
deparaffinization technique, as described in Example 1. After
deparaffinization the
biological samples were placed in a pressure cooker (Model #62104 Nordic Ware,
Minneapolis, MN) containing 1.5 L lx citrate buffer. The pressure cooker was
then
sealed and placed in a microwave oven (Model #MQS0836E, Matsushita, Franklin
Park, IL). With the microwave oven set on "high," the samples were subjected
to
microwave heating for approximately 30 minutes. After microwaving the samples
were
then "cured" for 30 minutes in the pressure cooker with the lid securely
fastened. After
curing the biological samples were placed in lx citrate buffer for two
minutes. The
biological samples were then removed from the citrate buffer and the end of
the slides
blotted to removed excess citrate buffer. After blotting, the slides were
placed on the
automated instrument and immunohistochemically stained as described above.
The biological samples deparaffinized by the solvent technique and by the
automated exposing and simultaneous cell conditioning technique were compared
after
immunohistochemical staining. Morphology on all sets of slide was acceptable
and
essentially equivalent.
Example 3
Two Step Automated "Exposing" and "Cell Conditioning"
Biological sample samples of tonsil T998D, tonsil Ki67, E68, E7, E33, E8, E29,
E15, and E68 that had been preserved in paraffin and treated with formaldehyde
were
treated by the following protocol:
Exposing and CeII Conditioning
1. Incubate for 2 minutes
2. Increase thermofoil temperature to 65.0° C
3. Incubate for 6 minutes
4. Rinse slide and apply coverslip
5. Incubate for 6 minutes
6. Rinse slide and apply coverslip
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7. Increase thermofoil temperature to
100.0 C
8. Adjust slide volume and apply liquid
coverslipTM
9. Rinse slide
10. Adjust slide volume and apply liquid
coverslipTM
11. Incubate for 4 minutes
12. Adjust slide volume and apply liquid
coverslipTM
13. Incubate for 4 minutes
14. Adjust slide volume and apply liquid
coverslipTM
15. Incubate for 4 minutes
16. Adjust slide volume and apply liquid
coverslipTM
17. Incubate for 4 minutes
18. Adjust slide volume and apply liquid
coverslipTM
19. Incubate for 4 minutes
20. Adjust slide volume and apply liquid
coverslipTM
21. Incubate for 4 minutes
22. Adjust slide volume and apply liquid
coverslipTM
23. Incubate for 4 minutes
24. Adjust slide volume and apply liquid
coverslipTM
25. Incubate for 4 minutes
26. Adjust slide volume and apply liquid
coverslipTM
27. Incubate for 4 minutes
28. Rinse slide
29. Decrease temperature to 42.0 C
30. Adjust slide volume and apply liquid
coverslipTM
31. Incubate for 4 minutes
32. Rinse slide
33. Decrease temperature to 20.0 C
34. Adjust slide volume and apply liquid
coverslipTM
35. Incubate for 4 minutes
36. Rinse slide
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The buffer used in the protocol was SSC buffer with either 20% formamide or
0.1% Triton. After the biological sample was subjected to the above protocol,
the DAB
paraffin protocol used for immunohistochemical staining of Example 2 was
applied.
Tonsil biological sample was treated with anti-Ki67 as a primary antibody.
Samples
E68, E7, E33, and E8 biological sample was treated with anti-estrogen receptor
(6F11)
as a primary antibody. E29, E15, and E68 biological sample was treated with
anti-
progesterone receptor (lA6) as a primary antibody.
Control biological samples were deparaffinized by a traditional solvent-based
de-paraffinization technique, as described in Example 1. After
deparaffinization the
biological samples were placed in a pressure cooker (Model #62104 Nordic Ware,
Minneapolis, MN) containing 1.5 L lx citrate buffer. The pressure cooker was
then
sealed and placed in a microwave oven (Model #MQS0836E, .Matsushita, Franklin
Park, IL). With the microwave oven set on "high", the samples were subjected
to
microwave heating-for approximately 30 minutes. After microwaving the samples
were
then "cured" for 30 minutes in the pressure cooker with the lid securely
fastened. After
curing the biological samples were placed in lx citrate buffer for two
minutes. The
biological samples were then removed from the citrate buffer and the end of
the slides
were blotted to removed excess citrate buffer. After blotting the slide were
placed on
the automated instnunent and immunohistochemically stained as described above.
The biological samples deparaffinized by solvent technique and by the
automated heating technique were compared after immunohistochemical staining.
Morphology on all sets of slide was acceptable and essentially equivalent.
Example 4
Automated Cell Conditioning of Non Paraffin Embedded Cell Lines for in situ
(Thin PrepsTM)
Hela (lot # 980427H), Caski (lot # 980416C) and Siha (lot # 9804165) cell
lines
stored in Cytyk* preparation solution (lot # 01139Q) were deposited on
microscope
slides using the Cytyk* 2000 instrument. After deposition the slides were
placed in
alcohol to keep moist until use on the Discovery~ In-Situ staining module
(Ventana
Medical Systems Inc., Tucson, AZ). Slides were loaded into the instrument and
wetted
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with 2 X SSC made from 20 X SSC (Ventana P/N 650-012). Slides were run through
a
cell conditioning protocol currently referred to as Depar 30 where the slides
are rinsed
with 2 X SSC and the temperature of the slides is increased to 95° C
for a period of
approximately 30 minutes. The slides are then cooled to 37° C and
rinsed with APK
Wash~ prior to the in-situ staining run.
Using the protocol Blue Swap ISH the cell lines were stained for HPV 16/18
(Enzo HPV 16/18 Bio Probe cat # 32874). Prior to probe application the cell
lines are
enzymatically digested with Protease* 2 (Ventana P/N 250-2019). After the
probe
application the probe and biological sample are denatured simultaneously at
95° C for 8
minutes. The non-specifically bound probe is washed off with stringency washes
of 2 X
SSC at 55° C. The probe is then detected with Streptavidin Alk Phos and
NBTBCIP.
The cell lines were dehydrated after staining with a one-minute exposure to 95
ethanol and a one-minute exposure to 100% ethanol repeated 2 times. Following
the
ethanol the slides were exposed to xylene for 3 minutes twice. After
dehydration the
slides were coverslipped.
The stained cell lines after conditioning showed acceptable morphology, there
was high background on these slides indicating a need for the process to be
developed
more.
Wet Load Slides
1. Skip Application & Incubate for 2 minutes
2. Rinse Slides (2X SSC Buffer) (Warm Slides to 65° C)
3. . Adjust Slide Volume, then Apply Coverslip
4. Skip Application & Incubate 6 minutes
5. Rinse Slides (2X SSC Buffer) (Warm Slides to 95° C)
6. Adjust Slide Volume, then Apply Coverslip
7. Rinse Slides
8. Adjust Slide Volume, then Apply Coverslip
9. Skip Application & Incubate for 4 minutes
10. Adjust Slide Volume, then Apply Coverslip
11. Skip Application & Incubate for 4 minutes
12. Adjust Slide Volume, then Apply Coverslip
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13. Skip Application & Incubate for 4 minutes
14. Adjust Slide Volume, then Apply Coverslip
15. Skip Application & Incubate for 4 minutes
16. Adjust Slide Volume, then Apply Coverslip
17. Skip Application & Incubate for 4 minutes
18. Adjust Slide Volume, then Apply Coverslip
19. Skip Application & Incubate for 4 minutes
20. Adjust Slide Volume, then Apply Coverslip
21. Skip Application & Incubate for 4 minutes
22. Rinse Slides (2X SSC Buffer) (Warm Slides to 37° C)
23. Adjust Slide Volume, then Apply Coverslip
24. Skip Application & Incubate for 4 minutes
25. Rinse Slides (APK Wash)
26. Adjust Slide Volume, then Apply Coverslip
Example 5
Automated "Exposing" and "Cell Conditioning"for single copy DNA detection
Slides containing formalin fixed, paraffin embedded cell lines Caski (R96-
lOSOA) and Siha (R96-96-C2) were stained on Ventana target slides. Slides were
dry
loaded onto the instrument and the slide temperature was increased to
65° C. Depar 30
protocol was run where the slides are rinsed with 2x SSC Buffer while at
65° C then the
heat is increased to 95° C for about 40 minutes. The slides were then
cooled to 37° C
and rinsed with APK wash. At this time the following In Situ protocol was run:
In-Situ Protocol: Tubbs I
(Dako TBST # 3306 is substituted for Ventana APK Wash during non-probe
steps)
Protease Digestion: Protease 2, 4 minutes, 37 °C
Inhibitor Step: Ventana Inhibitor from DAB kit 32 minutes 37° C
Probe: Enzo HPV Bio Probe 16/18
Control Probe: Enzo HPV Bio Probe 6/11
Denaturation: 95°C, 8 minutes
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Hybridization: 37°C, 64 minutes
2 Stringency Washes 2XSSC, 60°C, 8 minutes each
3'd Stringency Wash 2XSSC, 37°C, 4 minutes
Probe Detection: Streptavidin HRPO (Dako GenPoint #KU620)
Amplification: Biotinyl Tyramide (Dako GenPoint # K0620)
Detection: Streptavidin HRPO (Dako GenPoint #K0620)
or
Streptavidin Alk Phos (Vector # SA5100)
Chromogen DAB (Dako Gen Point # K0620)
or
Ventana NBTBCIP (Kit P/N)
or
Ventana Naphthol / Fast Red (Kit P/I~
I S Example 6
Automated "Cell Conditioning " for Non para~n Embedded Samples
The protocol for DAB staining as described in Example 2 was used in this
Example.
The cell conditioning steps for these antibodies was done after using a Cytyk
2000~ instrument to make ThinPreps~ of cell lines. The ThinPreps~ were stained
using
antibodies to ER, PgR, Ki67, P53 on Ventana ES instruments, NexES instruments
and a
manual procedure (Cytyk, Inc.). A duplicate group of slides have been stained
on the
NesES Insitu module, allowing the cell conditioning steps to be performed by
automation.
Although the example stated above is specific to the Cytyk~ instrument and
staining of the ThinPreps~, the experience is not limited to that mode of
making
cytological preps.
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Example 7
"Cell conditioning" of frozen biological sample
Frozen tonsil blocks 297C and 297D were prepared by cutting six sections from
each block and placing the sample on microscope slides. Four slides from each
block
were placed on the DiscoveryTM Insitu module and put through protocol Depar
10.
Slides are dry heated to 6S° for 6 minutes then rinsed with O.1M EDTA
buffer
pH 8. After rinsing, the slide is incubated at 65° for 20 minutes.
Slides were then
cooled to 37° C and rinsed with APK Wash. Two slides from each block
were left
untreated as controls. Following the Depar 10 treatment two treated slides
from each
block and one untreated slide were stained for H & E as described in Example
1. Two
treated slides from each block and one untreated from each block are stained
for LCA.
Run outcomes: for both the H & E and antibody staining there was no staining
difference between the treated and untreated slides.
From the foregoing detailed description, it will be appreciated that numerous
changes and modifications can be made to the aspects of the invention without
departure
from the true spirit and scope of the invention. This true spirit and scope of
the
invention is defined by the appended claims, to be interpreted in light of the
foregoing
specification.
