MXPA01001728A - Removal of embedding media from biological samples and cell conditioning on automated staining instruments - Google Patents

Abstract

The present invention provides an automated for removing or etching embedding media by exposing the biological samples for use in histochemical or cytochemical staining procedures without the dependence on organic solvents. The method comprises a biological sample slide in contact with a thermal platform, with or without heat, and with or without a fluid, to facilitate removal or etching of the embedding media from the biological sample. The present invention also provides an automated method for cell conditioning biological samples wherein the cells are predisposed for access by reagent molecules for histochemical and cytochemical staining procedures. The method comprises a biological sample slide in contact with a thermal platform, with or without heat, and with or without a fluid, to facilitate molecular access to cells and cell constituents within the biological sample.

Description

REMOVAL OF MEANS OF INTERCHANGE OF BIOLOGICAL SAMPLES AND CELLULAR CONDITIONING WITH AUTOMATED INSTRUMENTS COLORATION BACKGROUND OF THE INVENTION The present invention relates to the method for removing media from intercalation of biological samples in automated instruments before immunohistochemistry (IHC), in situ hybridization (ISH) or other cytochemical or histochemical manipulations. The present invention also relates to a method for conditioning cells or tissues in a manner that increases the accessibility of several molecules to their respective targets and generally to improve tissue and cell readability.

PREVIOUS ART The diagnosis of the disease based on the interpretation of cell or tissue samples taken from a diseased organism that has expanded dramatically over the years. In addition to traditional histological staining techniques and immunohistochemical tests, in situ techniques such as in situ hybridization and in situ polymerase chain reaction are now used to aid in the diagnosis of disease states in humans. Therefore, there is a variety of techniques that can determine not only the morphology, but also the presence of specific macromolecules in cells and tissues. Each of these techniques requires the preparation of cellular samples or tissues that may include fixing the sample with chemicals such as an aldehyde (such as formaldehyde, glutaraldehyde), formalin substitutes, alcohols (such as ethanol, methanol, sodium propane) or intercalating the sample in inert materials such as paraffin, celloidin, agars, polymers, resins, cryogenic media or a variety of plastic intercalation media (such as epoxy and acrylic resins). Other sample tissues or cell preparations require physical manipulation such as freezing (frozen tissue section) or aspiration through fine needles (fine needle aspiration (FNA)). Regardless of the tissue or cell sample or its method of preparation or preservation, the object of the technology specialist is to obtain accurate, reproducible and legible results that allow the exact interpretation of the data. One way to provide accurate, reproducible and legible data is to prepare the tissue or cells in a way that optimizes the test results regardless of the technique used. In the case of immunohistochemistry and in situ techniques, this medium increases the amount of signals obtained from the specific test (antibody, DNA, RNA). In the case of histochemical staining, the medium can increase the intensity of the coloration or increase the contrast of the coloration. Without preservation, tissue samples rapidly deteriorate so that their use in diagnostics is strongly compromised after the removal of their host. In 1893, Ferdinand Blum discovered that formaldehyde could be used to preserve or fix the tissue so that this tissue could be used in histochemical procedures. The exact mechanisms by which formaldehyde acts in the fixation of tissues are not fully established, but involve the cross-linking of the reactive sites in 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 bonds 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 reactions occur depending on conditions such as formalin concentration, pH, temperature and duration of fixation (Fox et al., J. Histochem, Cytochem 33: 845-853 (1985)). Some antigens, such as gastrin, somatostatin and a-1-antitrypsin, can 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)). The loss of antigen immunoreactivity is most notable in antigenic epitopes that are discontinuous, i.e., amino acid sequences wherein the formation of the epitope depends on the confluence of portions of the sequence of proteins that are not continuous. The Antigen Retrieval ™ is a term that describes the attempt to "undo" the structural changes that tissue treatment with a crosslinking agent induces in antigens residing in that tissue. Although there are several theories that attempt to describe the mechanism of the Antigen Retrival ™ so that the loosening or breaking of the reticulations formed by formalin fixation, it is clear that the modification of the protein structure by formalin is reversible under such conditions as a high temperature heating. It is clear that several factors affect the Antigen RetrivalTM: heating, pH, molarity and metal ions in solution (Shi et al., J. Histochem, Cytoche 45: 327-343 (1997)). Microwave heating seems to be the most important factor for the recovery of antigens hidden by formalin fixation. Microwave heating (100 ° + 5 ° C) generally produces better results in the Antigen Retrieval ™ immunohistochemistry (AR-IHC). Different heating methods have been described for the recovery of antigens in IHC such as self-partition (Pons et al, Appl. Immunohistochem 3: 265-267 (1995); Ban falvi et al., J. Path 174: 223- 228 (1994)), pressure cooking (Miller &Estran, Appl. Immunohistochem.3: 190-193 (1995); Norton et al., J. Path. 173: 371-379 (1994)); water bath (Kawai et al., Path. Int. 44: 759-764 (1994)), microwave cooking plus pressure cooking in plastic (US Patent No. Taylor et al., (1995); Pertschunk 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 produce satisfactory results when microwave cooking in distilled water, many antigens require the use of stabilizers during the heating process. Some antigens have particular pH requirements so that adequate results will only be achieved in a reduced pH range. Currently, most Antigen Retrieval ™ solutions are used at a pH of about 6-8, but there is some indication that slightly more basic solutions may provide marginally superior results (Shi, et al., J. Histochem, Cytochem. : 327-343 (1997)). Although the chemical components of the Antigen RetrievalTM solution, including metal ions, may play a role as a possible co-factor in the microwave cooking process, so far, no single chemical has been identified as being essential and the best for the Antigen Retrieval ™. Many solutions and methods are routinely used for color enhancements. These may include but are not limited to distilled water, EDTA, urea, Tris, glycine, saline and citrate stabilizer. Solutions containing a variety of detergents (ionic or non-ionic surfactants) can also facilitate the improvement of coloration over a wide range of temperatures (from room temperature to over 100 ° C). In addition to the cell surface molecules that can be represented in the cell portion, other molecules of interest in IHC, ISH, and other histochemical and histochemical manipulations are located in the cell, often in the nuclear envelope. Some of these molecules suffer from molecular transformation when exposed to a fixative (coagulator or precipitate) such as formalin. Therefore with respect to these molecules it is desirable not only to overcome the binding effects but also to increase the penetrability of the cell to facilitate the interaction of the organic and inorganic compounds with the cell. Other tissue samples can not be subjected to crosslinking agents before the test, but the improved results with respect to these tissues are also important. There are a variety of methods without formalin to preserve and prepare histological and cytological samples. Examples of these methods include but are not limited to a) blood spots, cytospins ™, ThinPreps ™, touch preps, cell lines, Ficoll separations for lymphocytes and buffy coatings, etc., are routinely preserved in several ways including but not limited to to outdoor drying, alcohol fixation, aerosol fixatives and storage media such as glycerin / sucrose storage media, b) tissues and cells (fixed or non-fixed) can be frozen and subsequently subjected to various stabilization techniques such as as preservation, fixation and drying, c) tissues and cells can be stabilized in a number of non-crosslinking aldehyde fixatives, fixatives that do not contain aldehyde, alcohol fixatives, oxidizing agents, heavy metal fixatives, organic acids and transport media. One route to improve the test results is to increase the signal obtained from a given sample. In a general sense, the increased signal can be obtained by increasing the accessibility of a given molecule to its target. As in the case of the antigens found in the cell, the targets in the cell can be made more accessible by increasing the permeability of the cell thereby allowing a greater number of molecules in the cell, thus increasing the probability that the molecule will "find" its target. Said increased permeability is especially important for techniques such as ISH, in situ PCR, IHC, histochemistry and cytochemistry. Tissues and cells are also interspersed in a variety of inert media (paraffin, celloidin, OCT ™, agar gum, plastics or acrylics, etc.) to help preserve them for future analysis. Many of these inert materials are hydrophobic and the reagents used for cytological and histological applications are predominantly hydrophilic, for this the inert medium may need to be removed from the biological sample before the test. For example, tissue sections interspersed with paraffin are prepared for subsequent tests by removing the paraffin from the tissue section by passing the plate through various organic solvents such as toluene, xylene, limonene or other suitable solvents. Traditional dewaxing uses organic solvents that generally require the process to be performed in ventilated hoods. In addition, the use and disposal of these solvents increases the cost of the analysis and the risk of exposure associated with each tissue sample tested. Currently, there is no technique available for the removal of inert media from the sample tissue by direct heating of the plate in an automated mold. It is now possible to remove inert media from the sample tissue while conditioning the sample or cell tissue in a single-step automated coloration process. The methods of the present invention allow a) the automated removal of the intercalation media without the use of organic solvents, therefore the exposure of the cells and therefore reduction of time, cost and protection against risks b) automated cellular conditioning without automated removal of the intercalation media of the sample cell or tissue, c) a multi-stage automated process that exposes the cells, performs cellular conditioning and increases the permeability of histological and cytological specimens, thereby increasing the readability of the sample and improving the interpretation of the test data. The methods of the present invention can be used to improve the staining and readability of most histological and cytological samples used in conjunction with histological and cytological staining techniques.

SUMMARY OF THE INVENTION The present invention relates to an automated method for the exposure of biological samples for use in cytological testing procedures by removing the intercalation media without the use of organic solvents. The present invention also relates to an automated method for cellular conditioning, thereby improving the accessibility of molecules in biological samples. The present invention also relates to an automated method for simultaneous exposure and cellular conditioning of biological samples for histochemical and cytological applications.

DETAILED DESCRIPTION OF THE PREFERRED MODALITIES One embodiment of the present invention relates to the exposure of biological samples by removal of inert materials in which biological samples have been interspersed for preservation and support. In a preferred embodiment of the present invention, paraffin or other inert materials are removed from the biological samples by heating one end of the biological sample. This can be accomplished by the pro-contact heating of the microscope plate in which the biological samples have been placed. Other inert materials that can be removed from the intercalated biological samples include but are not limited to agar gums and cryogenic media. This process of removing the inert intercalation media or the chemical attack of the intercalation media is referred to as exposure. In a preferred method of the present invention, the biological sample interspersed in paraffin placed on the glass plate is first heated by the heating element. The heating element exposes heat at one end of the biological sample (such as the thermal pallets described in US Patent Application 09 / 259,240 incorporated herein by reference) into an automated coloring instrument (US Patent Application No. of series 08 / 995,052 filed on December 19, 1997 and the provisional US patent application with serial No. 60 / 076,198 filed on February 27, 1998, both incorporated herein by reference) so that the sample plate and the paraffin are mixed. Typically, the biological sample is placed on the upper surface of a plate (such as a glass plate). The plate is then placed on the upper part of the thermal platform so that the lower surface of the plate is in contact with the thermal platform. The thermal platform, via conduction, heats the lower portion of the plate. After heating the biological sample, the inert material can be removed from the plate by a fluid (such as a gas or a liquid). For example, the inert material can be rinsed with DI water and a surfactant. In another method of the present invention, a biological sample interspersed in paraffin is placed in a glass plate of the microscope and the microscope plate is placed in a heating element. A reagent is placed in the plate of the biological sample, the plate of the biological sample is subsequently placed at elevated temperatures that would allow mixing by means of a fluid (such as a gas or a liquid).

In a preferred embodiment of the present invention, the reagents are used in conjunction with the heating of the intercalated biological samples. Appropriate reagents may include but are not limited to ionized water, citrate stabilizer (pH 6.0-8.0), Hc1-Tris stabilizer (pH 6-10), phosphate stabilizer (pH 6.0-8.0), SSC stabilizer, APK Wash ™ , acid stabilizers or solutions (pH 1-6.9), basic stabilizers or solutions (pH 7.1-14); mineral oil, Norpar, canola oil and PAG oil. Each of these reagents can also contain ionic and non-ionic surfactants such as Triton X-100, Tween, Brij, saponin and sodium dodecyl sulfate. In one embodiment of the present invention, the temperature of the heating element is raised by exceeding 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 exceeding the melting point of the paraffin in which the biological sample is intercalated. In a preferred method of the present invention, the temperature is raised by exceeding 50 ° C to about 130 ° O In a method of the present invention, the length of time required for the melting of the inert material will vary in accordance with the temperature used and the intercalation material. Typically, in an automated system, a processor such as a microprocessor is used in conjunction with a memory. The amount of time and temperature required to fuse the paraffin is contained in a table contained in the specification. The biological sample intercalated in paraffin is subjected to high 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.

Another embodiment of the present invention relates to the exposure of biological samples without the removal of the inert materials in which the biological samples have been interspersed for preservation and support. In a preferred embodiment of the present invention, the biological samples are ready for testing by contact heating of the microscope plate in which the biological samples have been placed. Other inert materials that have not been removed from biological samples include but are not limited to celloidin intercalation media or plastics and / or other polymers and resins. In a preferred method of the present invention, the intercalated biological sample placed on the glass plate is first heated by the heating element. The heating element exposes heat at one end of the biological sample, so that by using the thermal platforms described in US patent application. 09 / 259,240 in an automated coloring instrument (patent applications of E.U.A. 08 / 995,052 and 60 / 076,198) so that the sample plate is dried. In another method of the present invention, an interleaved biological sample was placed on a glass microscope plate and the microscope plate was heated at one end (i.e., by placing the plate on a thermal platform). Subsequently a reagent was placed on the plate of the biological sample and the plate of the biological sample, with the reagent, is subsequently heated to a specific temperature (oscillating from room temperature to more than 100 ° C) and for a specific amount of time (ranging from 2 minutes to 12 hours). This will cause the chemical etching of the surface in the inert intercalation material and after which the pickling reagent can be removed from the plate by a fluid (such as a gas or liquid). As previously described, the amount of time and the specific temperature can be stored in the memory.

In the preferred method of the present invention, the reagents are used together with or without heating of the biological samples interspersed. Appropriate reagents may include, but are limited to, ionized water, citrate stabilizer (pH 6.0-8.0), HC1-Tris stabilizer (pH 6-10), phosphate stabilizer (pH 6.0-8.0), SSC stabilizer, APK Wash ™ , acid stabilizers or solutions (pH 1-6.9), basic stabilizers or solutions (pH 7.1-14), mineral oil, Norpar, canola oil and PAG oil. Each of these reagents may also contain ionic or non-ionic surfactants such as Triton X-100, Tween, Brij, saponin and sodium dodecylsulfate. In the method of the present invention, the temperature of the heating element is set at an appropriate level for the drying or chemical etching of the intercalated biological sample. For example, etching can be carried out with a basic solution of methanol sodium hydroxide (sodium methoxide) at temperatures ranging from room temperature to 37 ° W. In the method of the present invention, the length of time required to strip the inert material will vary in accordance with the temperature used and the intercalation material (celloidin intercalation media or plastics and / or other polymers and resins, etc.). In a preferred method of the present invention the intercalated biological sample is subjected to appropriate temperatures ranging from 2 minutes to 12 hours. The heating element used in the method of the present invention requires that sufficient contact be maintained between the surface on which the biological sample is placed and the heating element. A preferred embodiment of the present invention also comprises an automated method of cellular conditioning, concurrent with, subsequent to or independent of the chemical attack removal of the inert intercalation material from the biological sample. It has been found that heating the biological sample in an appropriate reagent (organic or inorganic) improves the accessibility of the reagent to the target molecule in the cell (protein, nucleic acid, carbohydrate, lipid, pigment or other small molecule). This process of improving the accessibility of the reagent (organic or inorganic) to the molecular target is referred to herein as cellular conditioning. In a method of the present invention, cellular conditioning is achieved while the biological sample is exposed as described above. In this method of the present invention, a biological sample is placed on a glass plate of a microscope and the microscope plate is heated on its side (i.e., by placing the plate on a thermal platform) on an automated coloring instrument (US Patent Applications 08 / 995,052 and 60 / 076,198). A reagent is placed in 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 allow the fusion or chemical attack of the inert material and allow the cellular conditioning of the biological sample to be subsequently colored using histological or cytological techniques. The reagents used for cell conditioning may be the same as those for the exposure of the intercalated biological sample. For example, for DNA targets, a cellular conditioning solution may be an EDTA solution; A common temperature setting can be 95 ° C for a duration ranging from 6-60 minutes. For the target proteins, a cell conditioning solution may be a boric acid stabilizer solution, a common temperature setting may be exceeding 100 ° C for an oscillating duration of 6-60 minutes. For RNA targets, a cellular conditioning solution can be an SSC solution; A common temperature setting can be 75 ° C for a duration ranging from 6-60 minutes. For histochemical reactions, such as an Eosin and Hematoxylin stain (H &E), a cellular conditioning solution can be treated with ionized water, a common temperature can range from 60-80 ° C for a duration of 6-30 minutes. A partial list of possible reagents appears in Analytical Moríology, Gu, ed., Eaton Publishing Co. (1997) in pp. 1-40. The solutions should generally be of molarity, pH and known composition. Sodium dodecyl sulfate (SDS), ethylene glycol is preferably added to the conditioning solution. In addition, metal ions or other materials can be added to these reagents to increase the effectiveness of cell conditioning. In another method of the present invention, cellular conditioning is achieved 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 plate of the microscope and the microscope plate is heated at one end (i.e., by placing the plate on a thermal platform) on an automated coloring instrument (Requests US Patent 08 / 995,052 and 60 / 076,198). In this method the intercalated biological sample placed on the glass plate is first heated by the heating element in an automated coloring instrument so that the sample on the plate dries and the intercalation material is mixed or dewatered and removed by the application of a fluid. Subsequent to the exposure of the biological sample, an appropriate reagent is applied to allow cellular conditioning of the biological sample to be subsequently colored using histological or cytological techniques. The reagents used for cell conditioning may be the same as those for the exposure of the intercalated biological sample. For example, for DNA targets, a cellular conditioning solution can be an SSC solution; A common temperature setting can be 95 ° C for a duration ranging from 6-60 minutes. For protein targets, a cell conditioning solution may be a Phosphate Stabilizer solution, a common temperature setting may be exceeding 100 ° C for a duration ranging from 6-60 minutes. For RNA targets, a cellular conditioning solution can be an SSC solution, a common temperature setting can be 75 ° C for a duration ranging from 6-60 minutes. For histochemical reactions, such as a Trichrome stain, a cellular conditioning solution can be Bouins; A common temperature setting can range from 60-80 ° C for a duration of 6-30 minutes. In another preferred method of the present invention, cellular conditioning is achieved without exposing the biological sample. In this method of the present invention, a biological sample is placed in a glass plate of the microscope and the microscope plate is placed in a heating element in an automated coloring instrument. A reagent is placed in the biological sample and the temperature of the heating element may or may not be increased. The cellular conditioning of the biological sample can be carried out before the coloration is carried out using the histological and cytological techniques. The reagents used for cell conditioning may be the same as those for the exposure of the intercalated biological sample. For example, for DNA targets, a cell-conditioning solution may be a solution of Sodium Citrate; A common temperature setting can be 90 ° C for a duration ranging from 6-60 minutes. For protein targets, a cell conditioning solution may be a urea solution; A common temperature setting may be exceeding 100 ° C for a duration ranging from 6-60 minutes. For all cells, a cell conditioning solution can be a methanol solution, a common temperature setting can be room temperature for a duration of 4-10 minutes. For histochemical reactions, such as a Fast Bacilli Acid (AFB) stain, a cell conditioning solution can be peanut oil, a common temperature setting can be 60-70 ° C for a duration of 30-60 minutes. The present invention also comprises the cellular conditioning of cytological preps such as fine needle aspiration spots (FNA), touch preps, Ficoll, Cytospins®, Thins Preps®, vaginal-cervical fluid spots, body or blood fluid films, etc., which are fixed with an aldehyde not intercalated 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 will be dried and others placed in cytological fixatives, which may include Saccomanno and carbowax reagents (organic or inorganic) among others. The cells are centrifuged or filtered or directly touched on a glass plate and stained in some cases (PAP's) or applied directly against the plate (touch preps). The term "biological sample" means any collection of cells (detached or in tissue) that can be mounted on a standard microscope glass plate including, without limitation, organ sections, tumor sections, body fluids, spots, frozen sections, blood, cytological preps, microorganisms and cell lines. The term "Colorize" means any chemical or biological entity that, when applied to molecules in the biological sample, presents the detectable molecules under microscopic examination. The colorations include, without limit, detectable nucleic acid tests, antibodies and other agents which, in combination or by themselves, result in a colored terminal product (by bright or fluorescent fields).

The following examples are presented for illustrative purposes only and are not limiting of the invention in any way. Those skilled in the art will recognize that variants in the following may be made without exceeding the spirit and scope of the invention. All patents, patent applications and other publications are incorporated herein in their entirety as a reference.

EXAMPLES Example 1 Automated "Exposition" and "Cellular Conditioning" with Biological Samples Colored with H & AND The biological samples, including the chest CHTN33, stomach 149G, brain, tonsils and kidney, which have been embedded in paraffin were exposed according to the following procedure: the plates containing the biological sample referred to above were placed in an automated instrument (Window Medical Systems, Inc. Tucson, AZ) and were subjected to the exposure protocol described below. Generally, the plates containing the biological samples interspersed in paraffin were heated to dryness at 65 ° C for six (6) minutes then rinsed with 1x citrate stabilizer, ionized water, 10 mM phosphate stabilizer (pH = 6.3) or stabilizer HCI-Tris (pH = 7.4) each containing 0.1% Triton X-100.

Exposure Protocol 1 1. Incubate for 2 minutes 2. Rinse the plate 3. Adjust the volume of the plate and apply the coverslip ™ 4. Incubate for 6 minutes 5. Rinse the plate 6. Adjust the volume of the plate and apply the liquid coverslip ™ 7. Increase the temperature to 65.0 ° C 8. Rinse the plate 9. Adjust the volume of the plate and apply the liquid coverslip ™ 10. Incubate for 4 minutes 11. Adjust the volume of the plate and apply the liquid coverslip ™ 12. Incubate for 4 minutes 13. Adjust the volume of the plate and apply the coverslip ™ 14 liquid. Incubate for 4 minutes 15. Rinse the plate 16. Decrease the temperature to 42.0 ° C 17. Adjust the volume of the plate and apply the coverslip ™ 18. incubate for 4 minutes 19. Rinse the plate 20. Decrease the temperature to 42.0 ° C 21. Adjust the volume of the plate and apply the coverslip ™ 22 liquid. Incubate for 4 minutes 23. Rinse the license plate After automated exposure, the biological sample was stained with hematoxylin and eosin by the following method. Plates were placed in hematoxylin 1 (Richard Alien Scientific, Kalamazoo, Ml) for 1.5 minutes and then rinsed with running ionizing water for one minute. The plates were then placed in acid alcohol clarifier (Richard Alien Scientific) for one minute and then rinsed with running ionizing water for one minute. The plates were then placed in bluish ammonia reagent for one minute (Richard Alien Scientific, Kalamazoo, Ml) and subsequently rinsed in running ionizing water for one minute. The plates were then rinsed in 95% ethanol and then placed in 2.5% Y-eosin (Richard Alien Scientific, Kalamazoo, Ml) for 2.5 minutes. The biological samples on the plates were dehydrated by exposing the biological sample in a 100% ethanol bath for one minute. This process was repeated three times after exposure of the biological sample to a xylene bath for three minutes, twice. After the dehydration stage, the biological sample was covered with a coverslip. The biological control samples were dewaxed by a traditional solvent-based dewaxing technique. The biological sample placed on the microscope plates and preserved in paraffin was completely immersed in a xylene bath for five minutes. The plates containing the biological sample were placed in a second xylene bath for five minutes. After the removal of the second xylene bath they were placed in a 100% ethanol bath for three minutes. The plates were placed in a second bath of 100% ethanol for three minutes and then placed in a 90% ethanol solution for two minutes. Subsequently the plates were placed in 80% ethanol for one minute followed by complete immersion in distilled water for one to three minutes. After deparaffination, 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 automatic heating technique were compared after staining by hematoxylin and eosin. The morphology in all the equipment was acceptable and essentially equivalent. The biological samples of the brain and tonsils that were exposed by the automatic heating method showed more intensified haematoxylin staining than deparaffinized biological samples using standard solvent techniques.

Example 2 Automated "Exposure" of Biological Samples with Simultaneous "Cellular Conditioning" Biological samples of kidney Q-10 and tonsils T998D that have been fixed in formalin and interspersed in paraffin were exposed in accordance with the protocol described in Example 1 After the automated exposure, the biological sample was subjected to the DAB paraffin protocol used for immunohistochemical staining. The protocol for DAB staining is described below: I. Incubate for 2 minutes 2. Rinse plate 3. Adjust plate volume and apply liquid coverslip ™ 4. Rinse plate 5. Adjust plate volume and apply liquid coverslip ™ 6. Rinse plate 7. Adjust the volume of the plate and apply the coverslip ™ liquid 8. Apply a drop of the inhibitor 9. incubate for 4 minutes 10. Adjust the volume of the plate and apply the coverslip ™ II liquid. Apply one drop of the main antibody 12. incubate for 32 minutes 13. Adjust the volume of the plate and apply the coverslip ™ 14. Apply a drop of Biotinylated Ig 15. incubate for 8 minutes 16. Rinse the plate 17. Adjust the volume of the plate and apply the coverslip ™ 18. Apply a drop of Avidin-HRPO 19. incubate for 8 minutes 20. Rinse the plate 21. Adjust the volume of the plate and apply the liquid coverslip ™ 22. Apply a drop of DAB and a drop of H2O2 DAB 23. incubate for 8 minutes 24. Rinse the plate 25. Adjust the volume of the plate and apply the liquid coverslip ™ 26. Apply a drop of Copper 27. incubate for 4 minutes 28. Rinse the plate The main antibody used for the kidney biological sample Q-10 was Anti-CD15 (Ventana Medical Systems, Inc. Tucson, AZ, Catalog No. 250-2504). The main antibody used for the T998D biological sample from the tonsils was Anti-CD45RO (Ventana Medical Systems, Inc. Tucson, AZ, Catalog No. 250-2563). The used DaB coloring equipment was obtained from Ventana Medical Systems, Inc. Tucson, AZ, Catalog no. 250-001. The biological control samples were dewaxed by a traditional solvent-based dewaxing technique, as described in Example 1. After dewaxing the biological samples were placed in a pressure cooker (Model # 62104 Nordic Ware, Minneapolis, MN) containing citrate stabilizer 1.5 L 1x. The pressure cooker was subsequently sealed and placed in a microwave oven (Model # MQSO836E, Matsushita, Franklin Park, IL). With the microwave oven placed "high" the samples were subjected to microwave heating for approximately 30 minutes. After the microwaves were applied, the samples were subsequently "cured" for 30 minutes in the pressure cooker with the edge firmly fixed. After the cure of the biological samples, they were placed in a 1x citrate stabilizer for two minutes. The biological samples were subsequently removed from the citrate stabilizer and from the end of the stained plates in the citrate stabilizer of the excess removed. After staining, the plates were placed in the automated instrument and immunohistochemically colored as described above. The biological samples dewaxed by the solvent technique and by automated exposure and cell conditioning technique were compared after immunohistochemical staining. The morphology of all plate establishments was acceptable and essentially equivalent.

Example 3 Automated Two-Stage "Exposure" and "Cell Conditioning" The biological sample samples of tonsils T998D, amygdala Ki67, E68, E7, E33, E8, E29, E15 and E68 that had been preserved in paraffin and treated with formaldehyde were treated by the following protocol: Exposure and Cell Conditioning 1. Incubate for 2 minutes 2. Increase the temperature of the thermal sheet to 65.0 ° C 3. Incubate for 6 minutes 4. Rinse the plate and apply coverslip 5. Incubate for 6 minutes 6 Rinse the plate and apply coverslip 7. Increase the temperature of the thermal sheet to 100.0 ° C 8. Adjust the volume of the plate and apply the liquid coverslip ™ 9. Rinse the plate 10. Adjust the volume of the plate and apply the coversiip ™ liquid 11. Incubate for 4 minutes 12. Adjust the volume of the plate and apply the coversiip ™ liquid 13. Incubate for 4 minutes 14. Adjust the volume of the plate and apply the coversiip ™ liquid . Incubate for 4 minutes 16. Adjust the volume of the plate and apply the coversiip ™ liquid 17. Incubate for 4 minutes 18. Adjust the volume of the plate and apply the coversiip ™ liquid 19. Incubate for 4 minutes 20. Adjust the volume of the plate and apply the coversiip ™ liquid 21. Incubate for 4 minutes 22. Adjust the volume of the plate and apply the coversiip ™ liquid 23. Incubate for 4 minutes 24. Adjust the volume of the plate and apply the coversiip ™ liquid . Incubate for 4 minutes 26. Adjust the volume of the plate and apply the coversiip ™ liquid 27. Incubate for 4 minutes 28. Rinse the plate 29. Decrease the temperature to 42.0 ° C 30. Adjust the volume of the plate and apply the coversiip ™ liquid 31. Incubate for 4 minutes 32. Rinse the plate 33. Decrease the temperature to 20.0 ° C 34. Adjust the volume of the plate and apply the coversiip ™ liquid . Incubate for 4 minutes 36. Rinse the plate The stabilizer used in the protocol was SSC stabilizer with 20% formaldehyde or 0.1% Triton. After the biological sample was submitted under the above protocol, the DAB paraffin protocol used for the immunohistochemical staining of Example 2 was applied. Samples E68, E7, E33 and the E8 biological sample were treated with anti-estrogen receptor (6F11). ) as a main antibody. Samples E29, E15 and biological sample E68 were treated with the anti-progesterone receptor (1A6) as a primary antibody. The biological control samples were dewaxed by a traditional solvent-based dewaxing technique as described in Example 1. After dewaxing, the biological samples were placed in a pressure cooker (Model # 62104 Nordic Ware, Minneapolis, MN) containing citrate stabilizer 1.5 L 1x. The pressure cooker was subsequently sealed and placed in a microwave oven (Model # MQSO836E, Matsushita, Franklin Park, IL). With the microwave oven set to "high", the samples were subjected to microwave heating for approximately 30 minutes. After heating with microwaves, the samples were "cured" for 30 minutes in the pressure cooker with the edge firmly secured. After the cure of the biological samples, they were placed in 1x citrate stabilizer for two minutes. Subsequently, the biological samples were removed from the citrate stabilizer and the ends of the plates were stained to remove the excess citrate stabilizer. After staining of the plates, they were placed in the automated instrument and stained immunohistochemically as described above. The biological samples dewaxed by the solvent technique and by the automated heating technique were compared after immunohistochemical staining. The morphology of all plate establishments was acceptable and essentially equivalent.

EXAMPLE 4 Cellular Conditioning of Cell Lines Not Interleaved in Paraffin for in situ (Thin Preps ™) Cell lines Hela (lot # 9804271-1), Caski (lot # 980416C) and Siha (lot # 980416S) stored in the solution Cytyk preparation (batch # 01139Q) were deposited on the microscope plates using the Cytyk 2000 instrument. After deposition, the plates were placed in alcohol to maintain humidity until used in the Discovery® In-Situ staining module (Ventana Medical Systems Inc., Tucson, AZ). The plates were loaded on the instrument and moistened with 2X SSC made of 20 X SSC (Ventana P / N 650-012). The plates were run through a cell conditioning protocol currently referred to as Depar 30 where the plates were rinsed with 2X SSC and the temperature of the plates was increased to 95 ° C for a period of about 30 minutes. The plates were then cooled to 37 ° C and rinsed with APK Wash® before in situ coloration was run. Using the Blue Swap ISH protocol, the cell lines were stained with HPV 16/18 (Enzo HPV 16/18 Bio Test Cat # 32874). Before the application of the test, the cell lines were digested with Pase 2 (Ventana P / N 250-2019). After the application of the test, the test and the biological sample were denatured simultaneously at 95 ° C for 8 minutes. The non-specific binding test was washed with rigorous washes of 2X SSC at 55 ° O. The test was subsequently detected with Streptavidin Alk Phos and NBT / BCIP. The cell lines were dehydrated after staining with a one minute exposure with 95% ethanol and a one minute exposure with 100% ethanol repeated 2 times. After the plates were exposed to ethanol, they were exposed to xylene for three minutes twice. After dehydration the plates were covered.

The colored cell lines after conditioning showed acceptable morphology, there was a high antecedent in these plates indicating the need for the process to develop further. Loading Plates 1. Skip Application & Incubation for 2 minutes 2. Rinse the plates (Stabilizer 2X SSC) (Plates heated to 65 ° C) 3. Adjust the volume of the plate, then apply Coversiip 4. Skip the Application & Incubation for 6 minutes 5. Rinse the plates (2X SSC Stabilizer) (Plates heated to 95 ° C) 6. Adjust the volume of the plate, then apply Coversiip 7. Rinse the plates 8. Adjust the volume of the plate, then Apply Coversiip 9. Skip the Application & Incubation for 4 minutes 10. Adjust the volume of the plate, then apply Coversiip 11. Skip the Application & Incubation for 4 minutes 12. Adjust the volume of the plate, then apply Coversiip 13. Skip the Application & Incubation for 4 minutes 14. Adjust the volume of the plate, then apply Coversiip 15. Skip the Application & Incubation for 4 minutes 16. Adjust the volume of the plate, then apply Coversiip 17. Skip the Application & Incubation for 4 minutes 18. Adjust the volume of the plate, then apply Coversiip 19. Skip the Application & Incubation for 4 minutes 20. Adjust the volume of the plate, then apply Coversiip 21. Skip the Application & Incubation for 4 minutes 22. Rinse the plates (Stabilizer 2X SSC) (Plates heated to 37 ° C) 23. Adjust the volume of the plate, then apply Coversiip 24. Skip the Application & Incubation for 4 minutes 25. Rinse the Plates (Rinse APK) 26. Adjust the volume of the plate, then apply Coversiip Example 5"Exposure" and "Automated Cellular Conditioning" for the Detection of Single DNA Copy The plates containing the formalin-fixed paraffin-embedded cell lines Caski (R96-1050A) and Siha (R96-96-C2) were colored in the white plates Window. The plates were loaded dry in the instrument and the temperature was increased to 65 ° O The Depar 30 protocol was run where the plates were rinsed with 2x SSC Stabilizer at 65 ° C, then the heat was increased to 95 ° C by approximately 40 minutes The plates were subsequently cooled to 37 ° C and rinsed with APK rinse. At that time the following protocol was run In Situ: In-Situ Protocol: Tubbs 1 (Dako TBST # 3306 is replaced with the Window rinse during the stepless test) Protease digestion: Protease 2, 4 minutes, 37 ° C Stage Inhibitor: Inhibitor DAB device window 32 minutes 37 ° C Test: Enzo HPV Bio Test 16/18 Control test: Enzo HPV Bio Test 6/11 Denaturation: 95 ° C, 8 minutes Hybridization: 37 ° C, 64 minutes 2 Washes Rigorous 2XSSC, 60 ° C, every 8 minutes 3rd Rigorous Wash 2XSSC, 37 ° C, 4 minutes Test Detection: Streptavidin HRPO (Dako GenPoint # K0620) Amplification: Biotinyl Tiramide (Dako GenPoint # K0620) Detection: Streptavidin HRPO (Dako GenPoint # K0620) Streptavidin Alk Phos (Vector # SA5100) Cromogen DAB (Dako GenPoint # K0620) NBT / BCIP window (P / N equipment) Naphthol / Fast Red window (P / N equipment) Example 6 Automated "Cellular Conditioning" for Non-Interleaved Samples in Paraffin The protocol for DAB staining as described in Example 2 was used in this Example. The stages of cellular conditioning for these antibodies were performed after using a Cytik 2000® instrument to make ThinPreps® of the cell lines. ThinPreps® were stained using ER, PgR, Ki67, P53 antibodies in Ventana ES instruments, NexES instruments and a manual procedure (Cytyk, Inc.). A duplicate group of plates had been colored in the NesES Insitu module, allowing the cellular conditioning steps to be carried out by automation.

Although the example described above is specific for the Cytyk® instrument and the coloring of the ThinPreps®, the experience is not limited to the marking mode of the cytological preps.

Example 7"Cell Conditioning" of the frozen biological sample The frozen tonsil blocks 297C and 297D were prepared by cutting six sections of each block and placing the sample on the microscope plates. Four plates of each block were placed in the Discovery ™ Insitu module and put through the Depar 10 protocol. The plates are dried hot at 65 ° C for 6 minutes, then rinsed with 0.1 M EDTA stabilizer pH 8. After rinsing , the plate was incubated at 65 ° C for 20 minutes. The plates were subsequently cooled to 37 ° C and rinsed with Rinse APK. Two plates of each block were left untreated as the controls. After treatment with Depar 10, two plates from each block and an untreated plate were stained for H & E as described in Example 1. Two treated plates of each block and one untreated of each block were colored for LCA. The results of the operation: for the H & E and antibody staining there was no difference in staining between treated and untreated plaques. From the above detailed description, it will be appreciated that numerous changes can be made to aspects of the invention without departing from the spirit and scope of the invention. This scope and real spirit of the invention is defined by the accompanying claims to be interpreted in the light of the above specification.

Claims (64)

1. A method of removing media from intercalation of a biological sample, the method comprises the steps of: heating the biological sample and the intercalation medium and applying a fluid to the biological sample to separate the heated intercalation media from the biological sample.

2. The method according to claim 1, wherein the step of heating the biological sample and the intercalation means includes the melting of the intercalation media and wherein the application step includes flushing the fused intercalation media of the biological sample.

3. The method according to claim 1, wherein the biological sample is on an upper surface of a plate and wherein the heating step includes heating a lower end of the plate.

4. The method according to claim 3, wherein the lower end of the plate is in contact with a thermal platform and wherein the step of heating the lower end of the plate includes heating the plate by conduction using the thermal platform .

5. The method according to claim 4, wherein the step of heating a lower end of the plate includes heating the biological sample at temperatures ranging from ambient temperature to 130 ° C.

6. The method according to claim 1, wherein the intercalation medium is paraffin.

7. The method according to claim 1, wherein the fluid is a gas.

8. The method according to claim 1, wherein the fluid is a liquid.

9. The method according to claim 8, wherein the liquid is not an organic solvent.

10. The method according to claim 8, wherein the liquid is selected from the group consisting of ionized water, citrate stabilizer (pH 6.0-8.0), HCI-Tris stabilizer (pH 6-10), phosphate stabilizer (pH 6.0 -8.0), SSC stabilizer, APK Wash ™, acid stabilizers or solutions (pH 1-6.9), basic stabilizers or solutions (pH 7.1-14), mineral oil, Norpar, canola oil and PAG oil.

11. The method according to claim 1, wherein the fluid includes ionic and non-ionic surfactants.

12. The method according to claim 11, wherein the ionic and nonionic surfactants are selected from the group consisting of Triton X-100, Tween, Brij, sodium dylsulfate and saponin.

13. The method according to claim 1, wherein the fluid includes a detergent.

14. The method according to claim 1, further comprising the step of applying a liquid, the step of applying a liquid being carried out before the step of heating the biological sample.

15. The method according to claim 14, wherein the liquid is not an organic solvent.

16. The method according to claim 14, wherein the liquid includes water.

17. The method according to claim 16, wherein the liquid includes ionized water.

18. The method according to claim 1, wherein the steps of heating and rinsing are automated.

19. A method for removing the intercalation media from a biological sample, the method comprises the steps of: applying a liquid to a biological sample and mixing the intercalation media in the biological sample, the mixing step being carried out after the application step a liquid.

20. The method according to claim 19, wherein the liquid is not an organic solvent.

21. The method according to claim 19, wherein the liquid includes water.

22. The method according to claim 19, wherein the liquid is selected from the group consisting of ionized water, citrate stabilizer (pH 6.0-8.0), HCI-Tris stabilizer (pH 6-10), phosphate stabilizer (pH 6.0 -8.0), SSC stabilizer, APK Wash ™, acid stabilizers or solutions (pH 1-6.9), basic stabilizers or solutions (pH 7.1-14), mineral oil, Norpar, canola oil and PAG oil.

23. The method according to claim 19, wherein the liquid includes ionic and non-ionic surfactants.

24. The method according to claim 23, wherein the ionic and nonionic surfactants are selected from the group consisting of Triton X-100, Tween, Brij, sodium dodecylsulfate and saponin.

25. The method according to claim 19, wherein the liquid includes a detergent.

26. The method according to claim 19, wherein the steps of application and mixing are automated.

27. The method according to claim 19, wherein the mixing step of the intercalation means includes the application of heat to the biological sample.

28. The method according to claim 19, further comprising the step of applying a fluid to the biological sample to separate the mixed intercalation media from the biological sample.

29. The method according to claim 28, wherein the step of applying a fluid includes the rinsing of the mixed intercalation media of the biological sample with the fluid.

30. The method according to claim 28, wherein the fluid is not an organic solvent. 20. The method according to claim 19, wherein the liquid is not an organic solvent.

31. A method for removing the intercalation media from a biological sample, the method comprises the steps of: applying a liquid to a biological sample, the liquid not being an organic solvent and heating the biological sample.

32. The method according to claim 31, wherein the liquid is selected from ionized water, citrate stabilizer (pH 6.0-8.0), HCI-Tris stabilizer (pH 6-10), phosphate stabilizer (pH 6.0-8.0), SSC stabilizer, APK Wash ™, acid stabilizers or solutions (pH 1-6.9), basic stabilizers or solutions (pH 7.1-14), mineral oil, Norpar, canola oil and PAG oil.

33. The method according to claim 31, in the liquid includes ionic and nonionic surfactants.

34. The method according to claim 33, wherein the surfactants The monomers and nonionics are selected from the group consisting of Triton X-100, Tween, Brij, sodium dodecylsulfate and saponin.

35. The method according to claim 31, wherein the liquid includes a detergent.

36. The method according to claim 31, wherein the heating step includes mixing the intercalation media in the biological sample.

37. The method according to claim 36, further comprising the step of rinsing with a fluid the mixed intercalation media of the biological sample, the rinsing step being carried out after the mixing step.

38. An automated method of pickling media in a biological sample with a biological coloring process, the method comprises the steps of: applying at least one stripping solution and applying the fluid to remove at least one stripping solution.

39. An automated method according to claim 38, further comprising the step of applying heat to the biological sample after the step of applying at least one stripping solution.

40. An automated method according to claim 39, wherein the step of applying heat includes mixing the intercalation media.

41. An automated method according to claim 38, wherein the step of applying fluid to remove at least one stripping solution includes rinsing the plate with a rinsing means.

42. A method according to claim 41, wherein the rinsing medium is not an organic solvent.

43. An automated method according to claim 41, wherein the rinsing medium is selected from ionized water, citrate stabilizer (pH 6.0-8.0), HCI-Tris stabilizer (pH 6-10), phosphate stabilizer (pH 6.0- 8.0), SSC stabilizer, APK Wash ™, acid stabilizers or solutions (pH 1-6.9), basic stabilizers or solutions (pH 7.1-14), mineral oil, Norpar, canola oil and PAG oil.

44. An automated method according to claim 42, wherein the liquid includes ionic and nonionic surfactants.

45. An automated method according to claim 44, wherein the ionic and nonionic surfactants are selected from the group consisting of Triton X-100, Tween, Brij, sodium dodecylsulfate and saponin.

46. An automated method of cellular conditioning without the removal or chemical attack of the intercalation media in a biological coloring process, the method comprises the steps of: applying heat to the biological sample, applying at least one conditioning reagent and applying fluid to remove at least one conditioning reagent.

47. An automated method according to claim 46, wherein the biological sample is on the upper surface of a plate and wherein the heating step includes heating a lower end of the plate.

48. An automated method according to claim 47, wherein the lower end of the plate is in contact with a thermal platform and wherein the step of heating a lower end of the plate includes heating the plate by conduction using the thermal platform.

49. An automated method according to claim 46, wherein the biological sample is heated at temperatures ranging from room temperature to 130 ° W

50. An automated method according to claim 46, wherein at least one conditioning reagent is selected from the group consisting of ionized water, citrate stabilizer (pH 6.0-8.0), HCI-Tris stabilizer (pH 6-10), stabilizer of phosphate (pH 6.0-8.0), SSC stabilizer, APK Wash ™, acid stabilizers or solutions (pH 1-6.9), basic stabilizers or solutions (pH 7.1-14), mineral oil, Norpar, canola oil and PAG oil.

51. An automated method according to claim 46, wherein at least one conditioning reagent contains ionic and nonionic surfactants selected from the group consisting of Triton X-100, Tween, Brij, sodium dodecylisphate and saponin.

52. An automated method of simultaneous removal of the intercalation media from a biological sample while providing cellular conditioning in a biological coloring process, the method comprises the steps of: applying the cell conditioning and exposure reagents, applying heat to the sample biological, apply fluid to remove the reagents of cellular conditioning and exposure and coloration of the biological sample.

53. An automated method according to claim 52, wherein the biological sample is on the upper surface of a plate and wherein the heating step includes heating a lower end of the plate.

54. An automated method according to claim 53, wherein the lower end of the plate is in contact with a thermal platform and wherein the step of heating a lower end of the plate includes heating the plate by conduction using the thermal platform.

55. An automated method according to claim 52, wherein the step of applying heat includes heating the biological sample at temperatures ranging from room temperature to 130 ° W

56. An automated method according to claim 52, wherein the cell conditioning and exposure reagents are selected from the group consisting of ionized water, citrate stabilizer (pH 6.0-8.0), HCI-Tris stabilizer (pH 6). -10), phosphate stabilizer (pH 6.0-8.0), SSC stabilizer, APK Wash ™, acid stabilizers or solutions (pH 1-6.9), basic stabilizers or solutions (pH 15 7.1-14), mineral oil, Norpar, canola oil and PAG oil.

57. An automated method according to claim 52, wherein the cell conditioning and exposure reagents containing ionic and nonionic surfactants are selected from the group consisting of Triton X-100, Tween, Brij, 20 sodium dodecyl sulfate and saponin.

58. An automated method of removing or etching the intercalating media from a biological sample and subsequently providing the cellular conditioning in a biological coloring procedure, the method 25 comprises the steps of: applying heat to the biological sample; - apply a first fluid to the biological sample to remove the intercalation media or the etchant reagents, apply the cell conditioning reagents, apply a second fluid to remove the cell conditioning reagents 5 and the coloration of the biological sample.

59. An automated method according to claim 58, wherein the biological sample is on the upper surface of a plate and wherein the heating step includes heating a lower end of the plate.

60. An automated method according to claim 59, wherein the lower end of the plate is in contact with a thermal platform and wherein the step of heating a lower end of the plate includes heating the plate 15 by conduction using the thermal platform.

61. An automated method according to claim 58, wherein the step of applying heat includes heating the biological sample at temperatures ranging from room temperature to 130 ° C. 20

62. An automated method according to claim 58, further comprising the stage of applying exposure reagents to the biological sample.

63. An automated method according to claim 62, wherein the exposure reagents are selected from the group consisting of air, ionized water, citrate stabilizer (pH 6.0-8.0), HCI-Tris stabilizer (pH 6-10), phosphate stabilizer (pH 6.0-8.0), SSC stabilizer, APK Wash ™, acid stabilizers or solutions (pH 1-6.9), basic stabilizers or solutions (pH 7.1-14), mineral oil, Norpar, canola oil and PAG oil .

64. An automated method according to claim 62, wherein the exposure reagents containing ionic and nonionic surfactants are selected from the group consisting of Triton X-100, Tween, Brij, sodium dodecylisphate and saponin.