AU2006202931A1 - Automated immunohistochemical and in situ hybridization assay formulations - Google Patents
Automated immunohistochemical and in situ hybridization assay formulations Download PDFInfo
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- AU2006202931A1 AU2006202931A1 AU2006202931A AU2006202931A AU2006202931A1 AU 2006202931 A1 AU2006202931 A1 AU 2006202931A1 AU 2006202931 A AU2006202931 A AU 2006202931A AU 2006202931 A AU2006202931 A AU 2006202931A AU 2006202931 A1 AU2006202931 A1 AU 2006202931A1
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Classifications
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- G—PHYSICS
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- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
- G01N1/31—Apparatus therefor
- G01N1/312—Apparatus therefor for samples mounted on planar substrates
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
- G01N1/31—Apparatus therefor
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
- G01N35/00029—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor provided with flat sample substrates, e.g. slides
- G01N2035/00039—Transport arrangements specific to flat sample substrates, e.g. pusher blade
Description
S&F Ref: 648438D1
AUSTRALIA
PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT Name and Address of Applicant Actual Inventor(s): Address for Service: Invention Title: Ventana Medical Systems, Inc., of 1910 Innovation Park Drive, Tucson, Arizona, 85737, United States of America Penny Towne Joseph Utermohlen Chad Wilkinson Paula Rogers Spruson Ferguson St Martins Tower Level 31 Market Street Sydney NSW 2000 (CCN 3710000177) Automated immunohistochemical and in situ hybridization assay formulations The following statement is a full description of this invention, including the best method of performing it known to me/us:- 5845c AUTOMATED IMMUNOBISTOCHEMICAL
AND
IN SITU HYBRIDIZATION ASSAY FORMULATIONS REFERENCE TO RELATED
APPLICATIONS
g€ This application is a continuation-in-part of U.S. App. No. 09/800,689, filed 1 March 7, 2001, which is a continuation-in-part of U.S. App. No. 09/721,096, filed r, November 22, 2000, with Atty. Docket No. 98,541-C. U.S. App. No. 09/721,096 claimed the benefit of priority from International Application No. PCT/US99/20353, filed Sep. 3, C 10 1999, which was published on Mar. 16, 2000 as WO 00/14507. International Application No. PCT/US99/20353, in turn, claimed the benefit of priority from U.S. Provisional Patent Application No. 60/099,018, filed Sep. 3, 1998; U.S. Patent Application No.
09/259,240, filed Feb. 26, 1999; and International Application No. PCT/US99/04181, filed Feb. 26, 1999, which was published on Sep. 2, 1999 as WO 99/44030. U.S. Patent App. No. 09/721,096, filed November 22, 2000; International Patent App. No.
PCT/US99/20353, filed September 3, 1999, and published as WO 00/14507 on March 16, 2000; U.S. Provisional App. No. 60/099,018, filed September 3, 1998; U.S. Patent App.
No. 09/259,240, filed February 26, 1999 and International Patent App. No.
PCT/US99/04181, filed February 26, 1999, and published as WO 99/44030 on September 2, 1999, are all hereby incorporated by reference.
BACKGROUND OF THE INVENTION Field of the Invention The present invention relates to solutions methods, and instruments 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 of biological samples on automated instruments prior to immunohistochemical (IHC), in situ hybridization (ISH) or other histochemical or cytochemical manipulations.
Summary of the Related Art All patents, patent applications and non-patent articles or references mentioned 0 herein are hereby incorporated by reference to the extent that they are not contradictory.
The diagnosis of disease based on the 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 Ns used to help diagnose disease states in humans. Thus, there are a variety of techniques Sthat can assess not only cell morphology, but also the presence of specific macromolecules within cells and tissues.
For example, the diagnosis of breast, ovarian and other carcinomas may be facilitated by the use of techniques designed to identify the presence or absence of the cerb2/HER-2/neu protooncogene or the protein(s) expressed therefrom. The c-erb2/HER- 2/neu protooncogene is a member of the epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases. Amplification and overexpression of the c-erb2/HER-2/neu protooncogene is found in about 30% of breast carcinomas, about 20% of ovarian carcinomas and others. (Andrechek, E.R. et al., Proc. Natl. Acad. Sci. USA, vol. 97, no.
7, pp. 3444-3449 (2000); Doherty, J.K. et al., Proc. Natl. Acad. Sci. USA, vol. 96, pp.
10869-10874 (1999); Oh, J.J. et al., Nucleic Acids Res., vol. 27, no. 20, pp. 4008-4017 (1999); Klapper, L.N. et al., Proc. Natl. Acad. Sci. USA, vol. 96, pp. 4995-5000 (1999); and references found in each of the aforementioned articles.) Each of these techniques requires that sample cells or tissues undergo preparatory procedures that 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 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 j 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, etc.). In the case of histochemical staining it may mean increasing the intensity of S 5 the stain or increasing staining contrast.
C Without preservation, tissue samples rapidly deteriorate such that their use in Ci diagnostics is compromised shortly after removal from their host. In 1893, Ferdinand 0 Blum discovered that formaldehyde could be used to preserve or fix tissue so that it could CN 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 ca-l-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, 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 retrieval refers to 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 retrieval loosening or breaking of crosslinkages fomaed by formalin fixation), it is clear that modification of protein structure by formalin is reversible under conditions such as high-temperature heating. It is also clear that several factors affect Santigen retrieval: heating, pH, molarity and metal ions in solution (Shi et al., J.
Histochem. Cytochem. 45: 327-343 (1997)).
O Microwave heating appears to be the most important factor for retrieval of antigens masked by formalin fixation. Microwave heating (1000 5 0 C) generally yields C 5 better results in antigen retrieval immunohistochemistry (AR-IHC).
C Different heating methods have been described for antigen retrieval in IHC such C as autoclaving (Pons et al, Appl. Immunohistochem. 3: 265-267 (1995); Bankfalvi et al., J.
SPath. 174: 223-228 (1994)); pressure cooking (Miller Estran, Appl. Immunohistochem.
CN1 3: 190-193 (1995); Norton et al., J. Path. 173: 371-379 (1994)); water bath (Kawai et al., o0 Path. Int. 44: 759-764 (1994)); microwaving plus plastic pressure cooking Patent No. 5,244,787;; 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 microwave heating is perfonned 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 retrieval 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 retrieval solution, including metal ions, may play a role as possible co-factors in the microwave heating procedure, thus far, no single chemical has been identified that is both essential and best for antigen retrieval.
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 histochemical 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 O 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 CO 5 the cell.
o Other tissue samples may not have been subjected to cross-linking agents prior to C testing, but improved results with respect to these tissues is also important. There are a 0variety of non-formalin methods for preserving and preparing cytological and histological C, samples. Examples of these methods include, but are not limited to: a) hematology smears, cytospinsTM, ThinPrepsTM, touch preps, cell lines, Ficoll separations, etc. are routinely preserved in many ways which include, but are not limited to, air-drying, alcoholic fixation, spray fixatives and storage mediums such as sucrose/glycerin b) tissues and cells (either fixed or unfixed) may be frozen and subsequently subjected to various stabilizing techniques which include, but are not limited to, 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 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, OCTrM, 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; therefore, 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. These organic solvents are very volatile causing a variety of problems including requiring special processing deparaffminization is performed in ventilated hoods) and requires special waste disposal.
_The use of these organic solvents increases the cost-of analysis and exposure risk n 5 associated with each tissue sample tested and has serious negative effects for the environment.
n 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 C 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 multi-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.
SUMMARY OF THE INVENTION The present invention relates to solutions, methods and instruments for cell or tissue conditioning, which improves the accessibility of molecules in biological samples during histochemical or cytochemical assays. The conditioning may be performed at any point in a histochemical or cytochemical protocol.
The present invention further relates to an automated method for exposing biological samples for use in histological or cytological testing procedures by removing the embedding media without the use of organic solvents.
The present invention also relates to an automated method for the simultaneous exposing and cell conditioning of biological samples for histochemical and cytochemical applications.
BRIEF DESCRIPTION OF THE DRAWINGS O FIG. 1 is a perspective view of an instrument in which the formulations of the present invention may be used shown with the slide hood open and the carousel door 5 removed.
SFIG. 2 is a perspective view of an instrument in which the formulations of the Spresent invention may be used shown in conjunction with a computer and other N instruments with which it operates.
FIG. 3 is an exploded view of an instrument in which the formulations of the present invention may be used.
FIG. 4 is a perspective view of an instrument in which the formulations of the present invention may be used shown with a reagent dispenser.
FIG. 5 is a block diagram of the heating system of an instrument in which the formulations of the present invention may be used.
FIG. 6 is a flow chart for one embodiment for removing the embedding media from a slide.
FIGS. 7a-b are flow charts for one embodiment for cell conditioning.
DETAILED DESCRIPTION OF THE INVENTION The present invention provides reagents, formulations, aqueous compositions of matter and the like for use in an automated environment for cell conditioning of biological samples wherein the cells or tissues are predisposed for access by reagent molecules for histochemical and cytochemical staining procedures. The reagents comprise components optimized to facilitate molecular access to cells and cell constituents within the biological sample. The present invention also provides reagents for use in an automated environment for removing or etching embedding media by exposing a biological sample to be stained in histochemical or cytochemical procedures without the dependence on organic solvents. The reagents comprise components optimized to facilitate removal or etching of the embedding media from the biological sample.
One embodiment in which the formulations of the present invention may be useful relates to the exposing of biological samples by removal of the inert materials in which biological samples have been embedded for preservation and support. In a preferred embodiment, paraffin or other inert materials are removed from biological samples by M 5 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 in which the formulations of the present invention may be useful, 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 application 09/259,240, herein incorporated by reference) within an automated staining instrument patent application Serial No. 08/995,052 filed on December 19, 1997 and U.S. provisional patent application Serial No. 60/076,198 filed on February 27, 1998, both of which are herein incorporated by reference) such that the sample slide is dried and the paraffin is melted.
Heating Elements As discussed in U.S. patent application 09/259,240 (which is incorporated by reference) and referring now in detail to the drawings wherein like parts are designated by like reference numerals throughout, there is illustrated in FIG. 1 a perspective view of the molecular pathology apparatus in which formulations according to the present invention are useful, such apparatus is designated generally by reference numeral 10. Apparatus is designed to automatically stain or otherwise treat tissue mounted on microscope slides with nucleic acid probes, antibodies, and/or reagents associated therewith in the desired sequence, time and temperature. Tissue sections so stained or treated are then to be viewed under a microscope by a medical practitioner who reads the slide for purposes of patient diagnosis, prognosis, or treatmnent selection.
In a preferred embodiment of an apparatus in which the formulations of the present invention are useful, apparatus 10 functions as one component or module of a Ssystem 12 (FIG. which also comprises a host computer 14 preferably a personal computer, monitor 16, keyboard 18, mouse 20, bulk fluid containers 22, waste container 23 and related equipment. Additional staining modules or other instruments may be _added to system 12 to form a network with computer 14 functioning as a server.
Alternatively, some or all of these separate components could be incorporated into apparatus 10 making it a stand-alone instrument.
\The preferred configuration of apparatus 10 as well as system 12 is generally as Sdescribed in U.S. patent application serial no. 08/995,052 filed December 19, 1997 as r well as in the Ventana NexES User's G ide available from Ventana Medical Systems, Inc. (Tuscon, AZ), both incorporated herein by reference, except with respect to the novel heating system, slide support, bulk fluids module, volume adjust, and slide wipe as disclosed below. For purposes of clarity, detailed descriptions of those components found in both the present invention and the incorporated references are omitted.
In brief, apparatus 10 is a microprocessor controlled staining instrument that automatically applies chemical and biological reagents to tissue mounted on standard glass microscope slides. A carousel supporting radially positioned glass slides is revolved by a stepper motor to place each slide under one of a series of reagent dispensers. Apparatus 10 controls dispensing, washing, mixing, and heating to optimize reaction kinetics. The computer controlled automation permits use of apparatus 10 in a walk-away manner, i.e. with little manual labor.
More particularly, apparatus 10 comprises a housing formed of a lower section removably mounted or hinged to an upper section 32. A slide carousel 34 is mounted within lower section 30 for rotation about axis A-A. As set forth in greater detail below, a plurality of thermal platforms 50 are mounted radially about the perimeter of carousel 34 upon which standard glass slides with tissue samples may be placed. Carousel 34 is preferably constructed of stainless steel. It is a key feature of the present invention that the temperature of each slide may be individually controlled by means of various sensors and microprocessors as described herein. Also housed within apparatus 10 (FIG. 3) are wash dispense nozzles 36, CoverslipTM dispense nozzle 37, fluid knife 38, wash volume adjust nozzle 39, bar code reader mirror 40, and air vortex mixers 42 the details of which are discussed hereinafter.
Rotatably mounted atop upper section 32 is a reagent carousel 28. Dispensers 26 r- are removably mounted to reagent tray 29 (FIG. 4) which, in turn, is adapted to engage carousel 28. Reagents may include any chemical or biological material conventionally applied to slides including nucleic acid probes or primers, polymerase, primary and secondary antibodies, digestion enzymes, pre-fixatives, post-fixatives, blocking agents, readout chemistry, and counterstains. Reagent dispensers 26 are preferably bar code.
\0 labeled 29 for identification by the computer. For each slide, a single reagent is applied and then incubated for a precise period of time in a temperature-controlled environment.
Mixing of the reagents is accomplished by compressed air jets 42 aimed along the edge of the slide thus causing rotation of the reagent. After the appropriate incubation, the reagent is washed off the slide using nozzles 36. Then the remaining wash buffer volume is adjusted using the volume adjust nozzle 39. CoverslipTM solution, to inhibit evaporation, is then applied to the slide via nozzle 37. Air knife 38 divides the pool of Coverslip T M followed by the application of the next reagent. These steps are repeated as the carousels turn until the protocol is completed.
In addition to host computer 14, apparatus 10 preferably includes its own microprocessor 44 to which information from host computer 14 is downloaded. In particular, the computer downloads to microprocessor 44 both the sequence of steps in a run program and the sensor monitoring and control logic called the "run rules" as well as the temperature parameters of the protocol. Model No. DS2251T 128K from Dallas Semiconductor, Dallas TX is an example of a microprocessor that can perform this function.
Turning now to FIG. 5 there is shown a block diagram of the slide heating system 48. The system generally comprises about twenty thermal platforms 50, radially mounted to carousel 34, for heating the slides and monitoring the temperature thereof, and control electronics printed circuit board 52 also mounted to the slide carousel for monitoring the sensors and controlling the heaters. Control electronics 52 are mounted under the rotating slide carousel 34. Information and power are transfenrred. from the fixed instrument platform to the rotating carousel via a slip ring assembly 56. This information includes the temperature parameters needed for heating the slides (upper and lower) communicated from microprocessor 44 (after having been downloaded from computer 14) to control electronics 52 as described below. If, during a run, the slide temperature is determined to be below the programmed lower limit, the thermal platform heats the slide.
Likewise, if the slide temperature is found to be above the upper limit, heating is stopped.
O A power supply of sufficient capacity to provide about eight watts per heater is provided to meet the requisite rate of temperature rise (a/k/a "ramp up").
Similarly, in an alternate embodiment, the cooling of the slides may be likewise controlled, as described subsequently. In one alternate embodiment, cooling platforms "1 are mounted below the slide. The cooling platforms may comprise Peltier-type thermal Stransducers. In an alternative embodiment, a cooling device such as a fan (not shown) may optionally be provided if rapid cooling of the slides is required for particular applications. The cooling device will modify the ambient air for all of the platforms, necessitating the heaters corresponding to the slides which should not be cooled to compensate for the drop in ambient air temperature. The slide heating system described herein uses conduction heating and heats slides individually. The system provides more accurate on-slide temperature and allows for temperature settings on a slide by slide basis.
Heating of the Slide Typically, the biological sample is placed on a top surface of a slide (such as a glass slide). The slide is then placed on top of the thermal platform 50, so that the bottom surface of the slide is in contact with the thermal platform. As discussed previously, the thermal platform 50, 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 de-ionized water and a surfactant. Surfactants useful in the invention include, but are not limited to, Chemphos TC-227S (Chemron Corp.), Colawet DOSS70 (Colonial Chemical, Inc., South Pittsburg, TN), Colawet DOSS70E (Colonial Chemical, Inc., South Pittsburg, TN), Colawet DOSS70PG (Colonial Chemical, Inc., South Pittsburg, TN), ESI-TERGE T 320 (CCP/Atofina, Kansas City, MO), Geropon T-77 (Rhodia, Inc.), Geropon AS-200 (Rhodia, Inc.), Hostapon KCG (Clarient Corp.), Monafax 057 (Uniquema/ICI Surfactants), and the like. Preferably, the surfactant is Chemphos TC-227S or Hostapon KCG. More preferably, the surfactant is sodium dodecylsulfate.
The current method for deparaffinization is markedly different from what was performed in the prior art. Prior art methods include: using organic solvents to dissolve the paraffin; or manually using heat and dissolving agents to dissolve the paraffin. In contrast, in one aspect of the current invention, the deparaffinization does not Sinvolve a chemical reaction that dissolves the paraffin. In particular, the fluid which is placed on the sample does not solvate the paraffin. Instead, the current method involve the melting of the paraffin from the tissue and the washing of it away with fluids. In one embodiment, the fluid which is placed on top of the embedded sample is not miscible or O capable of being mixed with the paraffin. One example of this is water, which is not IN miscible with paraffin. In addition, water has a higher density than liquified paraffin.
SThe paraffin, when melted, floats to the top of the fluid so that the top of the slide may be rinsed, rinsing off of the melted paraffin.
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 Tris-HC1 buffer (pH 6-10), phosphate buffer (pH SSC buffer, APK WashTM, acidic buffers or solutions (pH 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. Other surfactants useful in the invention include, but are not limited to, Chemphos TC-227S (Chemron Corp.), Colawet DOSS70 (Colonial Chemical, Inc., South Pittsburg, TN), Colawet (Colonial Chemical, Inc., South Pittsburg, TN), Colawet DOSS70PG (Colonial Chemical, Inc., South Pittsburg, TN), ESI-TERGE T 320 (CCP/Atofina, Kansas City, MO), Geropon T-77 (Rhodia, Inc.), Geropon AS-200 (Rhodia, Inc.), Hostapon KCG (Clarient Corp.), Monafax 057 (Uniquema/ICI Surfactants), and the like. Preferably, the surfactant is Chemphos TC-227S or Hostapon KCG. More preferably, the surfactant is 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 S 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.
SIn a method of the present invention, the duration of time required to melt the inert Smaterial will vary according to the temperature used and the embedding material.
CTypically, 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 present invention requires that sufficient contact be maintained between the surface on which the biological sample is placed and the heating element.
Referring to Figure 6, there is shown a flow chart for one embodiment for removing the embedding media from a slide. As shown at block 60, the surface of the slide is agitated. In one embodiment, the vortex air mixers 42 are used to stir the biological sample on the surface of the slide. As shown at block 62, the temperature for the slide is raised to a predetermined temperature. In one embodiment, the temperature of the slides is raised by heating using the thermal platforms 50. As one example for in situ hybridization (ISH), the initial heating step heats the slide to 65 0 C for 16 minutes and then to 75 0 C for 2 minutes. In an in-munohistochenical (IHC) example, initial heating step raises the temperature of the thermal platforms to 75°C for approximately 4 minutes.
This initial heating step, while not necessary to remove the embedding medium, is performed in order to remove any moisture which is between the biological sample and the surface of the slide; and begin melting the embedding media (the melting point for paraffin is 50-57'C, as discussed previously).
The embedding media is both on the outside of the sample encasing the sample) and also infused in the tissue. When performing this initial heating, the embedding media may pool away from the tissue. Specifically, because the slide is at a slide angle (not completely horizontal), the embedding media may slowly pool at a point Son the slide which is lower than where the sample sits. In an alternate embodiment, the slide may be rinsed with a fluid (either liquid or gas) after the initial heating step to remove the embedding media that has pooled. For example, fluids such as de-ionized water may be used to rinse the embedding media from the slide.
S 5 As shown at block 64, an aqueous fluid is applied to the slide. Any aqueous fluid is acceptable such that the fluid covers the entire biological sample. As discussed previously, examples of aqueous fluid include de-ionized water, citrate buffer (pH Tris-HC1 buffer (pH 6-10), phosphate buffer (pH SSC buffer, APK C' Wash
TM
acidic buffers or solutions (pH basic buffers or solutions (pH 7.1-14), mineral oil, Norpar, canola oil, and PAG oil. Moreover, the aqueous fluid may also contain ionic or non-ionic surfactants such as Triton X-100, Tween, Brij, saponin and sodium dodecylsulfate. The surfactants lower the surface tension of the aqueous fluid, allowing the aqueous fluid to spread better over the surface of the slide. In one embodiment, the aqueous fluid comprises de-ionized water with about 0.1% Triton X- 100. Other surfactants useful in the invention include, but are not limited to, Chemphos TC-227S (Chemron Corp.), Colawet DOSS70 (Colonial Chemical, Inc., South Pittsburg, TN), Colawet DOSS70E (Colonial Chemical, Inc., South Pittsburg, TN), Colawet (Colonial Chemical, Inc., South Pittsburg, TN), ESI-TERGE T 320 (CCP/Atofma, Kansas City, MO), Geropon T-77 (Rhodia, Inc.), Geropon AS-200 (Rhodia, Inc.), Hostapon KCG (Clarient Corp.), Monafax 057 (Uniquema/ICI Surfactants), and the like. Preferably, the surfactant is Chemphos TC-227S or Hostapon KCG. More preferably, the surfactant is sodium dodecylsulfate.
An additional ingredient may be added, acting as an anti-microbial agent, so that the fluid prior to application on the slide does not contain microbes. In one embodiment, the fluid includes a water content, by weight, of 99% or greater the fluid is composed of between 99%-100% water). The use of water as a fluid to remove the embedding material is unlike what is conventionally used to remove the embedding material, such as organic solvents. Water is totally immiscible with paraffin, a typical embedding material. In contrast, organic solvents, such as toluene, xylene, limonene, are miscible with paraffin and therefore considered suitable for deparaffinization.
Further, the aqueous fluid should be applied in sufficient amounts and at sufficient times (accounting for evaporation of the aqueous fluid due to heating) such that the embedding media may float to the surface of the aqueous fluid and such that the biological sample on the slide will not dry out. In one embodiment, the aqueous fluid is applied sequentially, with a first application of approximately ImL of aqueous fluid on the biological sample, and with a second application two minutes later of aqueous fluid.
e C 5 The second application may be approximately .5mL to 1 mL of aqueous fluid. The fluid Smay be applied to the slide by using a nozzle which is position directly above the slide.
SIn this manner, the amount of fluid dropped onto the slide may be controlled. Moreover, Sbecause the fluid embedding material (such as paraffin) may have a low surface tension, 71 applying a stream of fluid onto the slide may not leave a sufficient amount of fluid on the top of the slide. Thus, using the nozzle to drop the fluid onto the embedded sample is preferred as it allows more of the fluid to remain on the upper surface of the slide. An additional consideration is the maintenance of the temperature of the biological sample above the melting point of the embedding media. In the paraffin example, the melting point is approximately 50-57°C. In a preferred embodiment, the sample is heated to 75°C. However, since the aqueous fluid is not heated, the application of the aqueous fluid to the slide lowers the temperature of the biological sample temporarily to approximately 60-65°C, which is still above the melting point of paraffin. Thus, the choice of temperature to heat the sample should be high enough so that the addition of fluids to the slide does not lower the temperature of the slide below the melting point of the embedding media. Alternatively, the fluid applied to the slide may be heated prior to application so as not to reduce the temperature of the sample on the slide.
At block 66, an evaporation inhibitor is applied. In a preferred embodiment, LIQUID COVERSLIPTM (LCS
TM
is applied. Thereafter, the system waits for a predetermined amount of time as the slide is heated, as shown at block 68. In in situ hybridization (ISH), one example of the time for heating the slide is 6 minutes at In an immunohistochemical (IHC) example, the incubation period is 8 minutes at 75 0
C.
In one embodiment, the temperature of the slides remains constant during the initial heating until step 76, as discussed subsequently. Thus, the heaters for the slides are turned on during the initial step of heating and remain on at the same temperature until after the embedding media is rinsed from the slide. Alternatively, the setpoint temperature of the heaters may be adjusted from the time of initial heating until after the embedding media is rinsed from the slide.
During the heating, the embedding media floats to the top of the aqueous fluid. In a preferred embodiment, the fluid which is applied to the slide has density which is O greater than the embedding media. In the specific example, the fluid is mostly composed of water 99% or greater) and the embedding media is paraffin. Paraffin, as an oil based product, is less dense than water. Thus, the paraffin rises to the top of the water after being melted.
The slide is then rinsed, as shown at block 70, carrying away the embedding Imedia in the aqueous fluid. In a preferred embodiment, the same type of aqueous fluid used to cover the slide is also used to rinse the slide. The rinse also leaves an amount of aqueous fluid on the biological sample. In a preferred embodiment, approximately mL is left on the slide. Thereafter, aqueous fluid is further applied to the slide, as shown at block 72. In an alternate embodiment, the rinsing of the slide may also leave a sufficient amount of aqueous fluid on the slide such that additional aqueous fluid need not be applied.
At block 74, evaporation inhibitor is applied, which in a preferred embodiment is Liquid CoverslipTM (LCSTM). Thereafter, the temperature of the slide is reduced, as shown at block 76. In a preferred embodiment, this is done by reducing or eliminating the heat applied by the thermal platform 50. Moreover, in a preferred embodiment, the temperature of the slide is reduced to a predetermined temperature of 42°C. This predetermined temperature is chosen such that all slides, when processing, are at a known temperature (as opposed to being at ambient temperature, which may fluctuate).
Thereafter, the apparatus waits a predetermined amount of time, as shown at block 78.
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 application 09/259,240 within an automated staining instrument patent applications 08/995,052 and 60/076,198) such that the sample O slide is dried.
In another method of the present invention, an embedded biological sample is S 5 placed on a glass microscope slide and the microscope slide is heated on one side 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 Sspecified temperature (ranging from ambient to greater than 100 0 C) and for a specified Samount of time (ranging from 2 minutes to 12 hours). This will cause etching of the 0o 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 Tris-HC1 buffer (pH 6-10), phosphate buffer (pH SSC buffer, APK WashTM, acidic buffers or solutions (pH basic buffers or solutions (pH7.1-14) mineral oil, Norpar, canola oil, and PAG oil. Each of these reagents may also contain ionic or non-ionic surfactants, which include, but are not limited to, Chemphos TC-227S (Chemron Corp.), Colawet DOSS70 (Colonial Chemical, Inc., South Pittsburg, TN), Colawet DOSS70E (Colonial Chemical, Inc., South Pittsburg, TN), Colawet DOSS70PG (Colonial Chemical, Inc., South Pittsburg, TN), ESI-TERGE T 320 (CCP/Atofina, Kansas City, MO), Geropon T- 77 (Rhodia, Inc.), Geropon AS-200 (Rhodia, Inc.), Hostapon KCG (Clarient Corp.), Monafax 057 (Uniquema/ICI Surfactants), and the like. Preferably, the surfactant is Chemphos TC-227S or Hostapon KCG. More preferably, the surfactant is sodium dodecylsulfate.
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 37C.
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 S(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 firom 2 minutes to 12 hours. The heating element used in the method of the present invention requires that sufficient contact be maintained E S between the surface on which the biological sample is placed and the heating element.
A preferred embodiment of the present invention also comprises an automated method of cell conditioning, either concurrent with, subsequent to or independent of removal or etching of the inert embedding material from the biological sample. Heating Sthe 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, etc.). This process of improving accessibility of the reagent (organic or inorganic) to the molecular target is referred to herein as cell conditioning.
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 by placing the slide on a thermal platform) within an automated staining instrument patent applications 08/995,052 and 60/076,198). 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.
Referring to Figures 7a-b, there are shown flow charts of one embodiment for exposing a biological sample to permit cell conditioning. As shown at block 80, it is determined whether cell conditioning is applied. A variety of cell conditioners may be applied depending on the processing necessary. As shown at block 82, the slide is rinsed with aqueous fluid. Any aqueous fluid is acceptable such that the fluid covers the entire biological sample. In a preferred embodiment, de-ionized water with Triton X-100 T M is used. Thereafter, an evaporation inhibitor is applied to the slide, as shown at block 84. In a preferred embodiment, Liquid Coverslip T is applied. Thereafter, the temperature of the slide is modified to a predetermined value, as shown at block 86. In a preferred Sembodiment, heaters heat the slide to 421C. This initial heating is performed in order to ensure that all of the slides begin the cell conditioning at a predetermined temperature; otherwise, the temperature of the slide may be unknown if the temperature of the slides is determined by the ambient temperature.
Evaporation inhibitor is then applied, as shown at block 88. Thereafter, the temperature of the slides is raised to a predetermined temperature, as shown at block ",1This is the temperature at which the cell conditioning is performed. In a preferred Sembodiment, the temperature of the heaters is set to 1 00 0 C. Thereafter, cell conditioner is applied, as shown at block 92 and evaporation inhibitor is applied, as shown at block 94.
A multitude of cell conditioners may be applied, as discussed herein.
How lightly/heavily a sample is fixed determines the amount of cell conditioning necessary. If a sample is lightly fixed, a mild cell conditioning is recommended.
Likewise, if the sample is moderately or heavily (extended) fixed, a moderate or a heavy cell conditioning, respectively, is recommended. Improper cell conditioning may have adverse consequences on the processing of the sample. In a preferred embodiment, cell conditioning mild/medium/ heavy time is 30/60/90 minutes respectively. Moreover, the cell conditioner is applied at predetermined increments within the processing in order to properly condition the cell and to avoid drying out the sample. As shown in Figure 7a, the number of times to iterate through the application of cell conditioner and LCS
T
is determined, as shown at block 96. This variable is called "loop_counter." Loopcounter is set to 0, as shown at block 98 in Figure 7b. A loop is entered (as shown at block 100) and the system waits a predetermined amount of time (as shown at block 102). In a preferred embodiment, the predetermined amount of time is 6 minutes. Cell conditioner is applied (as shown at block 104) and evaporation inhibitor is applied (as shown at block 106). The loop counter is then incremented, as shown at block 108. In a preferred embodiment, the number of times the loop is executed is 5/10/15 for mild/medium/heavy cell conditioning. Thereafter, the temperature of the slide is reduced, as shown at block 110. In a preferred embodiment, the heater temperature is reduced to 42 0
C.
Alternatively, the heater may be turned off and the slide temperature may revert to the ambient temperature. Thereafter, the slide is rinsed, as shown at block 112.
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 Smay be a solution of EDTA; a common temperature setting may be 95°C for a duration ranging from 2- 90 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 2- 90 minutes. For RNA targets, a cell conditioning solution 5 may be a solution of SSC; a common temperature setting may be 75°C for a duration C^ ranging from 2- 90 minutes. For histochemical reactions, such as a Hematoxylin and SEosin stain, a cell conditioning solution may be treated de-ionized water; a Scommon temperature may range from 60-80°C for a duration of 2- 90 minutes. A partial Clist 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) and/or 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 subsequent 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 by placing the slide on a thermal platform) within an automated staining instrument patent applications 08/995,052 and 60/076,198). In this method (one embodiment of which is shown in Figure 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 2- 90 minutes. For protein targets, a cell conditioning solution may be a solution of phosphate buffer; a common temperature setting may be in excess of 100°C for a duration ranging from 2- 90 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 2- 90 minutes. For histochemical reactions, such as a Trichrome stain, a cell conditioning solution may be Bouins; a common temperature may range from 0 80°C for a duration of 2-30 minutes.
In yet another method of the present invention, cell conditioning is accomplished 5 without the biological sample being exposed. In this method of the present invention, a Sbiological sample is placed on a glass microscope slide and the microscope slide placed Son a heating element within an automated staining instrument. A reagent is placed on the Sbiological 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 2-90 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 2-90 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).
The term "Biological sample" includes, but is not limited to, any collection of cells (either loose or in tissue) that can be mounted on a standard glass microscope slide Z including, without limitation, sections of organs, tumors sections, bodily fluids, smears, frozen sections, blood, cytology preps, microorganisms, tissue arrays and cell lines.
The term "Stain" includes, but is not limited to, any biological or chemical entity which, when applied to targeted molecules in biological sample, renders the molecules M 5 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).
SThe terms "Reagent", "Buffer", "Additive", "Component" and "Solution" as used CN herein for exposing or deparaffinizing the process of deparaffinization) may comprise the following component or components, all of which are available from Sigma Chemical, unless otherwise noted: de-ionized water, de-ionized water with about 0.1% Triton X-100, 10 mM phosphate at around pH 6.1, 10 mM phosphate with about 0.1% Triton X-100 at around pH 6.1, 10 mM citrate at around pH 6, 10 mM citrate with about 0.1% Triton X-100, 2xSSC, 10 mM Tris[hydroxymethyl]aminomethane chloride Tris-C1) at around pH 8.2, 10 mM Tris-C1 with about 0.1% Triton X-100 at around pH 8.2. A person skilled in the art to which this invention pertains will recognize that the concentration or concentrations of the component or components listed above may be varied without altering the characteristics of the reagent, buffer, additive or solution for exposing or deparaffinizing.
The terms "Reagent", "Buffer", "Additive", "Component", "Solution" and "Cell Conditioner" as used herein for cell conditioning may comprise the following component or components, all of which are available from Sigma Chemical, unless otherwise noted: mM citrate at around pH 6, 5 mM citrate with about 0.5% sodium dodecyl sulfate (SDS) at around pH 6, 10 mM citrate at around pH 6, 10 mM citrate with about SDS at around pH 6, 20 mM citrate at around pH 6, 20 mM citrate with about 0.5% SDS at around pH 6, 50 mM citrate at around pH 6, 50 mM citrate with about 0.5% SDS at around pH 6, 1 mM ethylene diamine tetraacetic acid (EDTA) at around pH 8, 1 mM EDTA with about 0.075% SDS at around pH 8, 10 mM EDTA at around pH 8, 10 mM EDTA with about 0.075% SDS at around pH 8, 20 mM EDTA at around pH 8, 20 mM EDTA with about 0.075% SDS at around pH 8, 50 mM EDTA at around pH 8, 50 mM EDTA with about 0.075% SDS at around pH 8, 10 mM citrate with about 0.5% SDS and about 1% ethylene glycol at around pH 6, 10 mM citrate with about 0.5% SDS and about ethylene glycol at around pH 6, 10 mM citrate with about 0.5% SDS and about ethylene glycol at around pH 6, 1 mM EDTA with about 0.075% SDS and about 1% ethylene glycol at around pH 8, 1 mM EDTA with about 0.075% SDS and about ethylene glycol at around pH 8, 1 mM EDTA with about 0.075% SDS and about ethylene glycol at around pH 8, phosphate/citrate/EDTA at about pH 9, 10 mM citrate Swith about 10 mM urea at around pH 6, 10 mM citrate with about 1 mM urea at around
C
pH 6.2, 10 mM sodium citrate with about 1.4 mM MgC1 2 and about 0.1% SDS at around pH 7, 10 mM sodium citrate with about 1.4 mM MgCI 2 and about 0.1% SDS at around CN pH 7.99, 10 mM Tris-Cl at around pH 8, 10 mM Tris-Cl with about 20% formamide at around pH 8, 10 mM citrate with about 5% dimethyl sulfoxide (DMSO) at around pH 6, mM citrate with about 0.1% Triton X-100 and about 20% formamide at around pH 6, mM phosphate with 5xSSC and about 2.5% chrondroitin A at around pH 7, 10 mM Tris-C1 with about 10 mM EDTA and about 0.1% Triton X-100 and about formamide at around pH 8.2, 10 mM citrate with about 20% glycerol at around pH 6, mM citrate with about 0.1% Triton X-100 and about 10 mM glycine at around pH 6, 1 mM EDTA with about 1 mM citrate and about 0.25% SDS at around pH 7.8, Norpar/mineral oil (high temperature coverslip), PAG-100 oil, 10 mM citrate with about 2% SDS at a pH of around 6 to around 6.2, 10 mM citrate with about 1% SDS at a pH of around 6 to around 6.2, 10 mM citrate with about 0.5% SDS at a pH of around 6 to around 6.2, 10 mM citrate with about 0.25% SDS at a pH of around 6 to around 6.2, 1 mM EDTA with about 2% SDS at a pH of around 7.5 to around 8, 1 mM EDTA with about 1% SDS at a pH of around 7.5 to around 8, 1 mM EDTA with about 0.5% SDS at a pH of around 7.5 to around 8, 1 mM EDTA with about 0.25% SDS at a pH of around to around 8, 1 mM EDTA with about 0.1% SDS at a pH of around 7.5 to around 8, 1 mM EDTA with about 0.075% SDS at a pH of around 7.5 to around 8, 0.5 mM EDTA with about 0.25% SDS at around pH 8, 10 mM EDTA with about 0.5% SDS at around pH 9.6.
A person skilled in the art to which this invention pertains will recognize that the concentration or concentrations of the component or components listed above may be varied without altering the characteristics of the reagent, buffer, additive or solution for cell conditioning.
A preferred embodiment of the present invention is the basic formulation comprising 10 mM Tris base, 7.5 mM boric acid, 1 mM EDTA (disodium salt), 0.05% SProClinTM 300 (Supelco, Inc., Bellefonte, PA) at pH 8,5. Other embodiments contemplated include the basic formulation wherein the Tris base concentration ranges 0 from about 5 mM to about 20 mM, wherein the boric acid concentration ranges from about 5 mM to about 40 mM, wherein the EDTA concentration ranges from about S 5 mM to about 2 mM or wherein the pH ranges from around pH 7 to around pH 9.
Temperature ranges contemplated are from around 95 0 C to around 100°C. Without intending to be construed as a limitation, it should be noted that no significant difference Sis seen when the concentration of Tris base or EDTA varies over the range specified above, significantly better results were obtained when the concentration of boric acid was 10 mM as opposed to 20 mM or 40 mM, slightly better results were obtained when the concentration of boric acid was 10 mM as opposed to 5 mM, conditioning is optimal at a pH of around 8 to around 8.6. Comparing 10 mM Tris 20 mM boric acid 1 mM EDTA 0.5% Brij 35 with 10 mM Tris 20 mM boric acid 0.5% Brij 35, both reagents displayed strongest conditioning at 100°C with significantly weaker conditioning at Other embodiments contemplated include, but are not limited to, citrate buffer (a combination of sodium citrate trisodium salt dihydrate and citric acid monohydrate hydrate), 10 mM Tris 20 mM boric acid 1 mM EDTA, 10 mM Tris 20 mM boric acid, 10 mM Tris 1 mM EDTA, 10 mM Tris, 20 mM boric acid, 1 mM EDTA, 20 mM Tris 20 mM boric acid 1 mM EDTA, 5 mM Tris 20 mM boric acid 1 mM EDTA, 10 mM Tris 20 mM boric acid 2 mM EDTA, 10 mM Tris 20 mM boric acid mM EDTA, 10 mM Tris 40 mM boric acid 1 mM EDTA, 10 mM Tris 10 mM boric acid 1 mM EDTA, 10 mM Tris 5 mM boric acid 1 mM EDTA, 10 mM Tris mM boric acid 1 mM EDTA, 10 mM Tris 20 mM boric acid 1 mM EDTA ethylene glycol, 10 mM Tris 20 mM boric acid 5% ethylene glycol, 10 mM Tris mM boric acid 1 mM EDTA 0.1% SDS, 10 mM Tris 20 mM boric acid 0.1% SDS, mM Tris 20 mM boric acid 1 mM EDTA 5% DMSO, 10 mM Tris 20 mM boric acid 5% DMSO, 10 mM Tris 20 mM boric acid 1 mM EDTA 10% DMSO, mM Tris 20 mM boric acid 10% DMSO, 10 mM Tris 20 mM boric acid 1 mM EDTA 5% formamide, 10 mM Tris 20 mM boric acid 5% formamide, 10 mM Tris 20 mM boric acid 1 mM EDTA 10% formamide, 10 mM Tris 20 mM boric acid formamide, 10 mM Tris 20 mM boric acid 1 mM EDTA 0.5% Brij 35, 10 mM Tris 20 mM boric acid 0.5% Brij 35, 10 mM Tris 20 mM boric acid 1 mM EDTA S+ 0.1% Brij 35, 10 mM Tris 20 mM boric acid 0.1% Brij 35, 10 mM Tris 20 mM boric acid 1 mM EDTA 0.5% Triton X-100, 10 mM Tris 20 mM boric acid Triton X-100.
The term "10 x SSC" refers to a 10 molar concentration of sodium C 5 chrloide/sodium citrate solution, comprising deionized water as needed to make a liter N solution, 87.66 g NaCI, 44.12 g citric acid trisodium salt, dihydrate, adjusted to pH C with HC1 or NaOH, as appropriate. 0.5 ml ProClin 300 is added as preservative. For all Sphosphate buffers prepared at any concentration (X molar), prepare X molar solutions of ciN HP0 4 using K 2
HPO
4 or NaHPO 4 and H 2 PO4 using KH 2
PO
4 or NaH 2
PO
4 Another preferred embodiment of the cell conditioner (cell conditioning solution 2 or CC2) comprises about 10 mM citrate buffer at about pH 6, about 5% ethylene glycol, about 1 mM sodium metabisulfite (MorphosaveTM, Ventana Medical Systems, Inc., Tucson, AZ; U.S. Patent No. 5,432,056) and about 0.3% sodium dodecyl sulfate (SDS).
The concentration of citrate may range from about 5 mM to about 50 mM. The pH of the buffer may range from about 4 to about 8. The concentration of ethylene glycol may range from about 1% to about 10%. The concentration of sodium metabisulfite may range from about 0.1 mM to about 10 mM, preferably from about 0.5 mM to about mM. The concentration of SDS may range from about 0.1% to about preferably from about 0.25% to about 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. All patents, patent applications, and other publications are hereby incorporated by reference in their entirety.
EXAMPLES
Example 1 Automated "Exposing" and "Cell Conditioning" with Biological Samples Stained with H&E Biological samples, including breast, stomach, 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 paraffin embedded biological samples were dry heated to 650 C for six minutes then rinsed with any of the following: 1) Ix citrate buffer, 2) de-ionized water, 3) 10mM phosphate buffer (pH or 4) 10mM Tris-HCI buffer (pH 7.4) each containing 0.1% Triton X-100.
Exposing Protocol 1 Incubate for 2 minutes Rinse slide Adjust slide volume and apply
COVERSLIPTM
Incubate for 6 minutes Rinse slide Adjust slide volume and apply LCSTM Increase temperature to 65.00 C Rinse slide Adjust slide volume and apply LCSTM Incubate for 4 minutes Adjust slide volume and apply LCSTM Incubate for 4 minutes Adjust slide volume and apply LCSTM Incubate for 4 minutes Rinse slide Decrease temperature to 42.00 C Adjust slide volume and apply LCSTM Incubate for 4 minutes Rinse slide Decrease temperature to 42.00 C Adjust slide volume and apply LCSTM LIQUID COVERSLIPLIQUID 22. Incubate for 4 minutes 23. Rinse slide After automated exposing, the biological sample was stained with hematoxylin 5 and eosin by the following method. Slides were placed in hematoxylin 1 (Richard Allen Ce¢ 0 Scientific, Kalamazoo, MI) for 1.5 minutes and then rinsed with running de-ionized water Sfor one minute. Slides were then placed in acid alcohol clarifier (Richard Allen k\ Scientific) for one minute and then rinsed with running de-ionized water for one minute.
O Slides were then placed in diluting ammonia-bluing reagent for one minute (Richard o0 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 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 deparaffinization technique. Paraffin-embedded biological samples placed on microscope slides and preserved in paraffin were completely submersed in a xylene bath for five minutes. Slides containing biological samples 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 deparaffmization, 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 Samples with Simultaneous "Cell Conditioning" c Biological samples of kidney and tonsil that had been formalin fixed and Sembedded in paraffin were exposed according to the protocol described in Example 1.
SAfter automated exposing, the biological sample was subjected to the automated DAB C 10 paraffin protocol used for immunohistochemical staining. The protocol for DAB staining is described below: DAB Protocol 1. Incubate for 2 minutes 2. Rinse slide 3. Adjust slide volume and apply LCSTM 4. Rinse slide Adjust slide volume and apply LCSTM 6. Rinse slide 7. Adjust slide volume and apply LCSTM S. Apply one drop of inhibitor 9. Incubate for 4 minutes Adjust slide volume and apply LCSTM 11. Apply one drop of primary antibody 12. Incubate for 32 minutes 13. Adjust slide volume and apply LCSTM 14. Apply one drop ofBiotinylated Ig Incubate for 8 minutes 16. Rinse slide 17. Adjust slide volume and apply LCSTM 18. Apply one drop of Avidin-HRPO 19. Incubate for 8 minutes Rinse slide 21. Adjust slide volume and apply LCS
T
22. Apply one drop of DAB and one drop DAB H202 23. Incubate for 8 minutes 24. Rinse slide 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 biological sample was (Ventana Medical Systems, Inc. Tucson, AZ, Catalogue no. 250-2504). The primary antibody used for the tonsil biological sample was Anti-CD45RO (Ventana Medical S 5 Systems, Inc. Tucson, AZ, Catalogue no. 250-2563). The DAB 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 C"1 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 #MQSO836E, 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 Ix 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 and staining on all sets of slides was acceptable and essentially equivalent.
Example 3 Two Step Automated "Exposing" and "Cell Conditioning" Biological samples of tonsil and breast that had been preserved in paraffin and treated with formaldehyde were treated by the following protocol: Exposing and Cell Conditioning Protocol 1. Incubate for 2 minutes 2. Increase thermofoil temperature to 65.00 C 3. Incubate for 6 minutes C 4. Rinse slide and apply LCSTM Incubate for 6 minutes 6. Rinse slide and apply LCSTM Cl 7. Increase thermofoil temperature to 100.00 C 8. Adjust slide volume and apply LCSTM 9. Rinse slide C 10. Adjust slide volume and apply LCSTM 11. Incubate for 4 minutes 12. Adjust slide volume and apply LCS T1 13. Incubate for 4 minutes 14. Adjust slide volume and apply LCS T M Incubate for 4 minutes 16. Adjust slide volume and apply LCSTM 17. Incubate for 4 minutes 18. Adjust slide volume and apply LCSTM 19. Incubate for 4 minutes Adjust slide volume and apply LCS T M 21. Incubate for 4 minutes 22. Adjust slide volume and apply LCSTM 23. Incubate for 4 minutes 24. Adjust slide volume and apply LCSTM Incubate for 4 minutes 26. Adjust slide volume and apply LCSTM 27. Incubate for 4 minutes 28. Rinse slide 29. Decrease temperature to 42.00 C Adjust slide volume and apply LCSTM 31. Incubate for 4 minutes 32. Rinse slide 33. Decrease temperature to 20.00 C 34. Adjust slide volume and apply LCSTM Incubate for 4 minutes 36. Rinse slide 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. Breast samples were treated with anti-estrogen receptor (6F11) or anti-progesterone receptor S(1A6) as a primary antibody. All primary antibodies are available through Ventana.
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 Ix citrate buffer. The pressure cooker was then Ssealed and placed in a microwave oven (Model #MQSOS36E, Matsushita, Franklin Park, SIL). With the microwave oven set on "high", the samples were subjected to microwave
C
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 instrument 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. Staining with Ki67 on all sets of slides was equivalent. ER (6F11) and PR (1A6) staining was slightly weaker with automated cell conditioning indicating the process does work but more development and optimization is required.
Example 4 Automated Cell Conditioning ofNon Paraffin Embedded Cell Lines for in situ hybridization (Thlin PrepsTM) Hela (ATCC lot 980427H), Caski (ATCC lot 980416C) and Siha (ATCC lot 980416S) 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 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 950 C for a period of approximately 30 minutes. The slides are then cooled to 370 C and rinsed with APK CO 5 Wash® prior to the in-situ staining run.
C Using the protocol Blue Swap ISH the cell lines were stained for HPV 16/18 C (Enzo HPV 16/18 Bio Probe cat 32874). Prior to probe application the cell lines are 0enzymatically digested with Protease 2 (Ventana P/N 250-2019). After the probe C application the probe and biological sample are denatured simultaneously at 950 C for 8 minutes. The non-specifically bound probe is washed off with stringency washes of 2 X SSC at 550 C. The probe is then detected with Streptavidin Alk Phos and NBT/BCIP.
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, weak staining and there was high background on these slides indicating a need for the process to be developed more.
Depar 30 Protocol Wet Load Slides 1. Skip Application Incubate for 2 minutes 2. Rinse Slides (2X SSC Buffer) (Warm Slides to 650 C) 3. Adjust Slide Volume, then apply LCSTM 4. Skip Application Incubate 6 minutes Rinse Slides (2X SSC Buffer) (Warm Slides to 950 C) 6. Adjust Slide Volume, then Apply LCSTM 7. Rinse Slides 8. Adjust Slide Volume, then Apply LCS T M 9. Skip Application Incubate for 4 minutes Adjust Slide Volume, then Apply LCSTM 11. Skip Application Incubate for 4 minutes 12. Adjust Slide Volume, then Apply LCSTM 13. Skip Application Incubate for 4 minutes 14. Adjust Slide Volume, then Apply LCSTM Z 15. Skip Application Incubate for 4 minutes S16. Adjust Slide Volume, then Apply LCSTM 17. Skip Application Incubate for 4 minutes 18. Adjust Slide Volume, then Apply LCS T M 19. Skip Application Incubate for 4 minutes Adjust Slide Volume, then Apply LCSTM 21. Skip Application Incubate for 4 minutes 22. Rinse Slides (2X SSC Buffer) (Warm Slides to 370 C) 23. Adjust Slide Volume, then Apply LCSTM c 10 24. Skip Application Incubate for 4 minutes N 25. Rinse Slides (APK Wash) 26. Adjust Slide Volume, then Apply LCSTM Example Automated "Exposing" and "Cell Conditioning"for single copy DNA detection Slides containing formalin fixed, paraffin embedded cell lines Caski (R96-1050A) and Siha (R96-96-C2), both generously provided by Dr. Raymond Tubbs, Cleveland Clinic Pathology Dept., Cleveland Ohio, were stained on Ventana target slides. Slides were dry loaded onto the instrument and the slide temperature was increased to 650 C.
The Depar 30 protocol was run wherein the slides are rinsed with 2x SSC Buffer while at 650 C then the heat is increased to 950 C for about 40 minutes. The slides were then cooled to 370 C and rinsed with APK wash. At this time the following in situ protocol was run: In situ Protocol: Tubbs 1 (Ventana APK Wash was used for all rinse 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 0 C, 8 minutes Hybridization: 37 0 C, 64 minutes 2 Stringency Washes 2XSSC, 60 0 C, 8 minutes each 3 rd Stringency Wash Probe Detection: Amplification: Detection: 2XSSC, 37°C, 4 minutes Streptavidin HRPO (Dako GenPoint Cat. #K0620) Biotinyl Tyramide (Dako GenPoint Cat. K0620) Streptavidin HRPO (Dako GenPoint Cat. #K0620)or Streptavidin Alk Phos (Vector Cat. SA5100) DAB (Dako Gen Point Cat. K0620) Chromogen Ventana NBT/BCIP (Kit P/N250-060) Ventana Naphthol Fast Red (Kit P/N250-030) Example 6 Automated "Cell Conditioning "for Non-paraffin 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 NexES 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.
I
Example 7 "Cell conditioning" of frozen biological sample Frozen tonsil blocks 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 in situ module and put through protocol Depar Slides are dry heated to 650 for 6 minutes then rinsed with 0.1M EDTA buffer pH 8. After rinsing, the slide is incubated at 650 for 20 minutes. Slides were then cooled to 370 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.
Example 8 Automated "Exposing" and "Cell Conditioning"for Immunohistochemistr y Various antibodies were assayed in the NexES Plus® (Ventana Medical Systems, Inc., Tuscon, AZ) automated environment according to the following protocol and flow chart: Protocol: (1) (2) (3) (4) (6) (7) (8) (9) Select EZ Prep Start Timed Steps If deparaffinization is selected Warm slide to 75°C and incubate for 4 minutes Adjust volume Apply LCSTM Incubate for 8 minutes Rinse slide Adjust volume Apply LCSTM (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) (25) (26) (27) (28) (29) (30) (31) (32) (33) (34) (35) (36) (37) (38) (39) (40) (41) (42) (43) (44) (45) (46) (47) (48) (49) (50) (51) (52) (53) (54) (55) (56) (57) Warm slide to 420C and incubate for 2 minutes If cell conditioning is selected If conditioner #1 is selected Rinse slide Adjust slide volume Apply LCST Warm slide to 42°C and incubate for 2 minutes Apply cell conditioning coverslip Warm slide to 100 0 C and incubate for 2 minutes Apply cell conditioner #1 Apply LCSTM If standard is selected Incubate for 6 minutes Apply cell conditioner #1 Apply LCS T M Repeat 9 times Warm slide to 420C and incubate for 2 minutes If mild is selected Incubate for 6 minutes Apply cell conditioner #1 Apply LCSTM Repeat 4 times Warm slide to 420C and incubate for 2 minutes If extended is selected Incubate for 6 minutes Apply cell conditioner #1 Apply LCSTM Repeat 14 times Warm slide to 420C and incubate for 2 minutes If conditioner #1 is not selected If conditioner #2 is selected Rinse slide Adjust slide volume Apply LCS T M Warm slide to 42 0 C and incubate for 2 minutes Apply cell conditioning coverslip Warm slide to 1000C and incubate for 2 minutes Apply cell conditioner #2 Apply LCSTM If standard is selected Incubate for 6 minutes Apply cell conditioner #1 Apply LCS
T
Repeat 9 times Warm slide to 42 0 C and incubate for 2 minutes If mild is selected Incubate for 6 minutes (58) (59) (61) (62) (63) (64) (66) (67) (68) (69) (71) (72) Apply cell conditioner #1 Apply LCSTM Repeat 4 times Warm slide to 42 0 C and incubate for 2 minutes If extended is selected Incubate for 6 minutes Apply cell conditioner #1 Apply LCSTM Repeat 14 times Warm slide to 42 0 C and incubate for 2 minutes Rinse slide Adjust slide volume Apply LCS T M Disable slide heater Select Reaction Buffer Table I summarizes the results from automated immunohistochemistry assays performed on the NexES Plus (also known as the BENCHMARKTM IHC) using the automated deparaffinization and cell conditioning protocols described herein.. The slides from the automated immunohistochemistry assays were compared to slides prepared using manual deparaffinization/cell conditioning protocols on a NexES instrument.
Specific antibody slides were prepared in triplicate. Negative control slides were prepared singly. Slides from both assays were analyzed by persons skilled in the art and assigned ratings for specific staining intensity and non-specific background staining intensity. The ratings for all of the slides from the NexES Plus fully automated assays exceeded the ratings for the manually deparaffinized and cell conditioned NexES slides in both specific staining intensity and non-specific background intensity. Slides were analyzed and rated by a minimum of two independent persons skilled in the art and average staining intensities were determined. The column marked "Qualified Tissue" in Table 1 represents the standard control tissue used to standardize the antibody used on it.
The standardized antibody is given a rating of 4.0. As can be seen in the column marked "Average Staining Intensity," most of the automated deparaffinized/cell conditioned slides met or exceeded those results.
2006202931 07 Jul 2006 TABLE 1: Antibody and Cell Conditioning Results Antibody Clone Qualified Pretreatment Incubation Nepative Average Neizative Tissue Time Control Stainin2 Control (minutes) Intensity anti-PR 1 A6 Endol I299#2 60 min. cell conditioning 32 NC Ig 4 0 anti-ER ON1 UT31698V 60 miii. cell conditioning 32 NC Ig 4 0 anti-Pan Keratin AE I, AE3, PCK26 SK1 97A 4 min. Protease 1 16 NC Ig 4 0 anti-C-erbB-2 CBI 1 TMC197G 60 min. cell conditioning 32 NC Ig 4 0 anti-S 100 polyclonal p19 None 16 Rbt NC .4 0 L26 T1398H None 16 NC Ig 3.9 0 anti-Vimentin 3B34 245A 8 min. Protease 2 16 NC Ig 4 0 anti-LCA RP2/18 T31099B None 16 NC Ig 4 0 anti-Melanosome HMB45 97-715C None 16 NC Ig 4 0 anfi-CEA TF-3H8-l CCA98157 None 16 NC Ig 4 0 A6 T9810163 None 16 NC Ig 3.6 0 anti-Chroniogratin LK2H1O pN None 16 NC Ig 4 0 2006202931 07 Jul 2006 TABLE 1: Antibody and Cell Conditioning Results (continued) _I
I
Atibod I Clone Qualified Tissue Pretreatment Incubation Time (minutes) Negative Control Average Staining Intensity Negative a,,ti-EMA Mc5 BRCA98139BC None 16 NC Ig 3.75 0 anti-Kappa Polyclonal T4282 4 min. Protease 1 16 Rbt NC 4 0 anti-Lambda Polyclonal 51099 4 min. Protease 1 16 Rbt NC 4 0 anfi-CD 15 MMA K121698 None 16 NC Ig 4 0 anti-Desmin DE-R- I1I SI1173B3C 16 min Protease 2 16 NC Ig anti-Muscle Actin HUCI-1 DIA8699A None 16 NC Ig 4 0 anti-PSA Polyclonal 271A None 16 Rbt NC 4 0 anti-Ki67 MM1 P92-3622 60 min. cell conditioning 16 NC Ig 3.9 0 anti-p53 bp-53- 11 EV246B 60 min. cell conditioning 16 NC Ig 4 0 anti-Keratin 5D3 L103CB 8 min Protease 1 16 NC Ig 4 0 aynti-NSE BBSINC/VI-H 14 #2 None 16 NC Ig 3.9 0 anti-bcl-2 bcl-2/1 00/D5 Block6 60 min. cell conditioning 32 NC Ig 4 0 2006202931 07 Jul 2006 TABLE 1: Antibody and Cell Conditioning Results (continued) Antibody Clone Qualified Pretreatment Incubation Nepative Averaize Negative Tissue Time Control Staining Control (minutes) Intensity anlti-CD3O IG12 H-odgkins1912696C 60 min. cell conditioning 16 NC Ig 3.75 0 anfi-CD43 L60 T11698H None 16 NC Ig 3.75 0 anti-GFAP Polyclonal CBR9872E None 16 Rbt NC 4 0 aini-Synaptophysin 27G 12 406A ND 16 ND 4 0 NOTE: In Table 1, ND stands for No Data.
111i pilllaly alltIUL&uiC davauIaoID Ic TOI vemlana IVI Icai .ys[ems, Inc., Luscon, O AZ) referred to in Table 1 (above) are described below, in alphabetical order: anti-bcl-2: Clone bcl-2/100/D5 is a monoclonal antibody used to detect bcl-2, which is an inhibitor of apoptosis. This antibody may be an aid to distinguish between reactive and neoplastic follicular proliferation. Control: Tonsil. (VMSI 760c 2693) CN anti-C-erbB-2 (HER2/neu): c 10 Clone CB11 is a monoclonal antibody to c-erb-B2, which is localized on the cell N membrane, and occasionally, in the cytoplasm of some neoplastic cells. (VMSI 760-2994) anti-CD Clone MMA is a monoclonal antibody used to aid in the identification of cells of the granulocytic lineage and/or Reed-Steinberg differentiation. Control: Hodgkin's Lymphoma. (VMSI 760-2504) Clone L26 is a monoclonal antibody used to aid in the identification of cells of the B lymphocytic lineage. Contol: Tonsil. (VMSI 760-2531/760-2137) anti-CD43: Clone L60 is a monoclonal antibody that specifically binds to antigens located in the plasma membrane of normal granulocytes and T lymphocytes. Control: Tonsil. (VMSI 760-2511) Clone X148 is a monoclonal antibody that specifically binds to antigens located in the plasma membrane of normal B lymphocytes and a subset of T lymphocytes.
Control: Tonsil. (VMSI 76-2510) Clone A6 is a monoclonal antibody that binds to the plasma membrane of cells of the T lineage and a subset of B-cells. Control: Tonsil. (VMSI 760-2563) anti-CEA: Clone TF-3H8-1 specifically binds to antigens located in the plasma membrane and cytoplasmic regions of mucosal epithelial cells. Control: Colon Carcinoma.
(VMSI 760-2507/760-2141) anti-Chromogranin: Clone LK2H10 is a monoclonal antibody that binds the chromogranin protein located in the secretory granules of normal and neoplastic neuroendocrine cells.
Control: Pancreas. (VMSI 760-2519/760-2140) Santi-Desmin: SClone DE-R-11 is a monoclonal antibody used to aid in the identification of cells of the myocytic lineage. Desmin is an intermediate filament found in mature smooth, striated and cardiac muscle. Control: Vas Deferens. (VMSI 760-2513) anti-EGFR: Clone 31G7 is directed against a transmembrane glycoprotein present on a variety of cells. (VMSI 760-2548) Santi-EMA: 0 Clone Mc5 is a monoclonal antibody used to aid in the identification of cells of epithelial lineage. EMA is of value in distinguishing both. large-cell anaplastic O carcinoma from diffuse histiocytic lymphoma, and small-cell anaplastic Scarcinoma from well and poorly differentiated lymphocytic lymphomas. Control: Carcinoma. (VMSI 760-2508) anti-ER: Clone 6F11 is a monoclonal antibody used to detect the presence of estrogen receptor. (VMSI 760-2596/760-2132) Clone CC4-5 is a monoclonal antibody also used to detect the presence of estrogen receptor. (VMSI 760-2546/760-2138) anti-GFAP: GFAP polyclonal antibody is directed against glial fibrillary acidic protein present in the cytoplasm of most human astrocytes and ependymal cells. This reagent may be used to aid in the identification of cells of the glial lineage. Control: Brain. (VMSI 760-2516) anti-Kappa: Kappa light chains are expressed by cells of the B-cell lineage. Light chain production by lymphoid cells is genetically restricted such that the immunoglobulin molecules produced by an individual cell will only contain a single light chain class. This clonal restriction may be used to indicate the polyclonal or monoclonal nature of B-cell and plasma cell populations. Control: Plasmacytoma. (VMSI 760-2514) anti-Keratin: Clone 5D3 is a monoclonal antibody raised against human epidermal keratins.
This antibody may be used to aid in the identification of cells of the epithelial lineage. This antibody reacts with cytokeratins 8 and 18. Control: Carcinoma.
(VMSI 760-2501) Clone AE1 is a monoclonal antibody raised against human epidermal keratins.
This antibody may be used to aid in the identification of cells of the ductal epithelial lineage. AE1 reacts with cytokeratins 10, 13, 14, 15 and 19. Control: Liver. (VMSI 760-2521) anti-Pan Keratin: c Pan Keratin is a cocktail of monoclonal antibody clones AE1/AE3/PCK26 used to aid in the identification of cells of the epithelial lineage. This antibody cocktail reacts with all cytokeratins except 9, 11 and 12. Control: Skin. (VMSI 760- 2595/760-2135) anti-Ki67: Clone MMI is a monoclonal antibody that specifically binds to nuclear antigens associated with cell proliferation that are present throughout the active cell cycle S(G1, S, G2 and M phases), but absent in resting (GO phase) cells. This reagent may be used to aid in the identification of proliferating cells. Control: Tonsil.
N (VMSI 760-2520) anti-Lambda: Lambda light chains are expressed by cells of the B-cell lineage. Light chain production by lymphoid cells is genetically restricted, such that the immunoglobulin molecules produced by an individual cell will only contain a single light chain class. This clonal restriction may be an aid to indicate the polyclonal or monoclonal nature of B-cell and plasma cell populations. Control: Plasmacytoma. (VMSI 760-2515) anti-LCA: Clone RP2/18 is a is a monoclonal antibody used to aid in the identification of cells of lymphocytic descent. This antibody specifically binds to antigens located predominantly in the plasma membrane and cytoplasmic rim of lymphocytes with variable reactivity to monocytes/histocytes and polymorphs. Control: Lymphoma.
(VMSI 760-2505/760-2136) anti-Melanosome: Clone HMB45 monoclonal antibody is used to aid in the identification of cells of the melanocytic lineage. It reacts with an antigen expressed in abnormal melanocytes and melanoma cells. Control: Melanoma. (VMSI 760-2518/760- 2139) anti-Muscle Actin: Clone HUC1-1 is a monoclonal antibody used to aid in the identification of cells of myocytic descent. Muscle actin is expressed in cells of the striated, smooth and cardiac muscle lineage. Control: Ileum. (VMSI 760-2502) anti-NSE: Clone BBS/NC/VI-H14 is a monoclonal antibody that reacts with an antigen that may be expressed in the cytoplasm of neurons, neuro-endocrine cells, endocrine tumors, carcinoids and Merkel cell tumors. Control: Pancreas. (VMSI 76-2517) anti-p53: Clone Bp-53-11 is a monoclonal antibody directed against the p53 protein. This Sreagent may be used to aid in the identification of abnormally proliferating cells in neoplastic cell populations. Control: Carcinoma. (VMSI 760-2540) anti-PCNA: Clone PCIO is a monoclonal antibody that may be used to aid in the identification of proliferating cells in various cell populations. The PCNA antigen is expressed in all proliferating cells in the G1, S, G2 and M phases. Control: Tonsil. (VMSI c, 760-2503) 10 anti-PSA: SPSA polyclonal antibody reacts with the secretory protein expressed in the cytoplasm of prostate epithelial cells. Control: Prostate. (VMSI 760-2506) anti-PSAP: Clone PASE/4LJ is a monoclonal antibody used to aid in the identification of cells of the prostate lineage. PSAP specifically binds to antigens located in the cytoplasmic regions of the normal prostate epithelial cells. Control: Prostate.
(VMSI 760-2509) anti-PR: Clone 1A6 is a monoclonal antibody used to aid in the identification of the progesterone receptor in human tissue. (VMSI 760-2547/760-2133) anti-S00: S100 polyclonal antibody is used to aid in the identification of cells of normal and abnormal neuro-endocrine descent. Control: Skin. (VMSI 760-2523/760-2133) anti-Vimentin: Clone 3B4 is used to aid in the identification of cells of mesenchymal origin.
Control: Vas Deferens. (VMSI 760-2512/760-2134) Negative Control: Polyclonal serum applied to negative tissue controls as part of quality control procedures for polyclonal antibodies. (VMSI 760-1023) Negative Control Ig: Clone MOPC-21 is applied to negative tissue controls as part of quality control procedures for monoclonal antibodies. (VMSI 760-2014) Example 9 Automated "Cell Conditioning" with or without "Exposing" for Immunohistochemistly (IHC), Imiunocytochemisty (ICC) or In situ Hybribidization
(ISH)
SThe CC2 solution may be used on automated immunohistochemical, immunocytochemical or in situ hybridization instruments BenchMark T M
ISH,
Ventana Medical Systems, Inc., Tucson, AZ; BenchMarkM IHC, Ventana Medical Systems, Inc., Tucson, AZ; etc.) with reagents designed to detect the c-erb2/HER-2lneu gene or protein(s) expressed therefrom INFORM® HER-2/neu DNA test, Ventana Medical Systems, Inc., Tucson, AZ; PATHWAY T M HER-2/neu (CB11) protein test, Ventana Medical Systems, Inc., Tucson, AZ; etc.).
For an ISH assay, the CC2 solution may preferably be used in an automated pretreatment step following deparaffinization with the CC2 solution applied to the tissue at about 90°C for about 10 minutes prior to application of the DNA probe. The heat treatment in conjunction with the CC2 solution allows the genomic DNA to become better suited for binding with the c-erb2/HER-2/neu DNA probe.
For an IHC assay, the CC2 solution may preferably be used in an automated cell conditioning step following deparaffinization with the CC2 solution applied to the tissue at about 95°C for about 30 minutes. The high temperature in conjunction with the CC2 solution allows the c-erb2/HER-2/neu protein (antigen or epitope) to become better suited for binding with the anti- c-erb2/HER-2/neu antibody.
These are representative contexts in which the CC2 solution is preferably used.
The CC2 solution is also useful in many other assays, techniques, protocols or procedures designed to detect the presence or absence of targeted DNA or protein molecules under consideration.
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.
Claims (16)
1. An aqueous composition of matter, comprising citrate buffer, ethylene glycol, sodium metabisulfite and a surfactant.
2. A composition according to claim 1, wherein the composition is buffered from approximately pH 4 to approximately pH 9. c 3. A composition according to claim 1, wherein composition is buffered at 0approximately pH 6.
4. A composition according to claim 1, wherein the composition comprises less than or equal to approximately 100 mM citrate buffer. A composition according to claim 1, wherein the composition comprises approximately 10 mM citrate buffer.
6. A composition according to claim 1, wherein the composition comprises less than or equal to approximately 10% ethylene glycol.
7. A composition according to claim 1, wherein the composition comprises approximately 5% ethylene glycol.
8. A composition according to claim 1, wherein the composition comprises less than or equal to approximately 10 mM sodium metabisulfite.
9. A composition according to claim 1, wherein the composition comprises 1 mM sodium metabisulfite. A composition according to claim 1, wherein the composition comprises less than or equal to approximately 10% SDS.
11. A composition according to claim 1, wherein the composition comprises approximately 0.3% SDS.
12. A composition according to claim 1, wherein the composition comprises approximately 10 mM citrate buffer at approximately pH 6, approximately 5% ethylene glycol, approximately 1 mM sodium metabisulfite and approximately 0.3% SDS.
13. An aqueous composition of matter comprising a buffer, a glycol, sodium metabisulfite and a surfactant.
14. A composition according to claim 13, wherein the buffer is a citrate buffer. A composition according to claim 14, wherein the buffer is at approximately pH 6. c
16. A composition according to claim 13, wherein the glycol is ethylene glycol.
17. A composition according to claim 13, wherein the concentration of the buffer is less than or equal to approximately 100 mM, the concentration of the glycol is less than or equal to approximately 10%, the concentration of the sodium metabisulfite is less than or equal to approximately 10 mM and the concentration of the SDS is less than or equal to approximately
18. A composition according to claim 17, wherein the buffer is citrate at a concentration of approximately 10 mM and the pH of the composition is approximately 6.
19. A composition according to claim 18, wherein the glycol is ethylene glycol and the concentration of ethylene glycol is approximately wherein the concentration of sodium metabisulfite is approximately 1 mM and wherein the concentration of SDS is approximately 0.3%. An aqueous composition of matter comprising approximately 10 mM citrate buffer at approximately pH 6, approximately 5% ethylene glycol, approximately 1 mM sodium metabisulfite and approximately 0.3% surfactant.
21. An aqueous composition of matter according to claim 20, wherein the surfactant is SDS. DATED this Seventh Day of July, 2006 Ventana Medical Systems, Inc. Patent Attorneys for the Applicant SPRUSON FERGUSON
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Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2011109580A1 (en) * | 2010-03-03 | 2011-09-09 | Battelle Memorial Institute | Sample preparation for spectroscopy analysis |
CN110132695A (en) * | 2019-05-27 | 2019-08-16 | 福州迈新生物技术开发有限公司 | A kind of pathological staining system reduction human error is caused to dye abnormal method |
US10436682B2 (en) | 2010-03-03 | 2019-10-08 | Battelle Memorial Institute | Sample preparation for spectroscopy analysis |
CN114594244A (en) * | 2022-01-26 | 2022-06-07 | 浙江莱阅病理诊断科技有限公司 | Low-pH antigen repairing buffer solution adapted to full-automatic immunohistochemical staining machine and preparation method thereof |
-
2006
- 2006-07-07 AU AU2006202931A patent/AU2006202931A1/en not_active Abandoned
-
2008
- 2008-10-17 AU AU2008229993A patent/AU2008229993A1/en not_active Abandoned
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2011109580A1 (en) * | 2010-03-03 | 2011-09-09 | Battelle Memorial Institute | Sample preparation for spectroscopy analysis |
US9034278B2 (en) | 2010-03-03 | 2015-05-19 | Battelle Memorial Institute | Sample preparation for spectroscopy analysis |
US9354146B2 (en) | 2010-03-03 | 2016-05-31 | Battelle Memorial Institute | Sample preparation for spectroscopy analysis |
US10436682B2 (en) | 2010-03-03 | 2019-10-08 | Battelle Memorial Institute | Sample preparation for spectroscopy analysis |
CN110132695A (en) * | 2019-05-27 | 2019-08-16 | 福州迈新生物技术开发有限公司 | A kind of pathological staining system reduction human error is caused to dye abnormal method |
CN114594244A (en) * | 2022-01-26 | 2022-06-07 | 浙江莱阅病理诊断科技有限公司 | Low-pH antigen repairing buffer solution adapted to full-automatic immunohistochemical staining machine and preparation method thereof |
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