| Deparaffinization compositions for dewaxing tissue specimens -> Monitor Keywords |
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  Deparaffinization compositions for dewaxing tissue specimensRelated Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Fixed Or Stabilized, Nonliving Microorganism, Cell, Or Tissue (e.g., Processes Of Staining, Stabilizing, Dehydrating, Etc.; Compositions Used Therefore, Etc.)Deparaffinization compositions for dewaxing tissue specimens description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20060019332, Deparaffinization compositions for dewaxing tissue specimens. Brief Patent Description - Full Patent Description - Patent Application Claims
TECHNICAL FIELD [0001] This application relates to compositions and methods for removal of wax from wax-embedded biological samples. BACKGROUND [0002] Paraffin has been used for many years as an embedding medium in the preparation of tissue specimens for sectioning in a microtome to produce specimen sections for histological studies. Such embedding processes generally include the well known steps of specimen fixation, dehydration, clearing, paraffin infiltration or impregnation, blocking or embedding in a block of paraffin, slicing the block and specimen into thin sections, mounting the sections on slides, removing the paraffin and solvents employed for this purpose (deparaffinizing), and staining the sections prior to microscopic analysis. The primary purpose of the embedding medium is to permit the specimens to be sectioned and mounted in the natural state. Plastic resins have also been used as embedding medium to provide a harder specimen that allows cutting of thinner sections. However, the use of paraffin-embedding has the advantage that the wax can be dissolved away from specimens prior to staining, allowing sections to be stained in the form of naked slabs of biopolymer and avoiding the extra difficulties and artifacts associated with the presence of unremovable resin-embedding medium (Horobin 1991). [0003] Recent improvements in paraffin-embedding compositions broaden its applicability while maintaining its compatibility with downstream manipulation and analysis of samples. For example, an improved paraffin-based embedding material, which includes a mixture of paraffin and an effective amount of ethylene-vinyl acetate copolymer (0.5% to 5% by weight of paraffin) allows shorter infiltration time and thinner sections (U.S. Pat. No. 4,497,792). Another improvement, the double-embedding technique, yields sections of thin tissue membranes, such as rodent mesenteric membranes that usually measure only 10 microns in thickness. In this method several membranes are fixed and mounted on four needles located at the bottom of a plastic box and then embedded in agarose. The agarose block is removed, dehydrated in alcohol, cleared with HistoPetrol (tradename for a mixture of isoparaffin hydrocarbons), permeated with paraffin and sectioned. The observed tissue morphology is comparable to that obtained with methacrylate plastic embedding but is less time-consuming, less hazardous since no plastic hardener and activator are used, and makes immunohistochemical studies easier (Ghassemifar et al. 1992). [0004] Consequently, deparaffinization of fixed, e.g. formalin fixed, paraffin embedded tissue sections is still a widely used methodology, particularly in hospital histopathology laboratories for immunodiagnostic purposes. [0005] Xylene, which is a flammable, volatile and toxic organic solvent, is currently commonly used in protocols to solubilize paraffin for deparaffinization of specimen sections. Typically, the microscope slide-mounted specimen is immersed in a xylene bath until the paraffin is solubilized. The deparaffinized specimen is then washed with a series of alcohol solutions of decreasing alcohol concentration, typically as baths in which the specimen is immersed, to remove xylene before a final wash with water. Efforts have been made to replace xylene in the deparaffinization process with less toxic and less volatile solvents. Terpene oil (e.g. available under the tradename AmeriClear from Baxter Health Care Diagnostics, Inc. McGaw Park, Ill.) and isoparaffinic hydrocarbons (e.g. available under the tradename Micro-Clear from Micron Diagnostics, Inc., Fairfax, Va.) produced equal deparaffinization compared to xylene (Jones et al. 1993). However, a series of alcohol washes were still required to remove either solvent prior to the water wash to achieve compatibility with most types of staining, particularly immunohistochemical staining. Furthermore, the use of paraffin-embedded specimens with automated systems, such as immunostainers, is increasing. [0006] Accordingly, there is still a need for deparaffinization compositions and methods that can effectively remove paraffin or improved paraffin-based embedding materials from specimens prior to histochemical or other diagnostic analyses, while minimizing danger to users, allowing compatibility with automated systems, and maintaining compatibility with downstream analyses. Deparaffinization compositions and methods that entail no or limited toxicity or carcinogenicity, produce no or minimal odors, reduce the quantity of toxic solvents used, minimize hazardous wastes, and/or decrease corrosiveness and flammability are needed. CITED LITERATURE [0007] 1. Horobin, R. W., In Histochemical and Immunochemical Techniques: Application to pharmacology and toxicology, (1991) Bach, P. and Baker, J., eds., Chapman & Hall, New York, N.Y. pp 1-9. [0008] 2. Ghassemifar, R. et al. (1992) "A double-embedding technique for thin tissue membranes" Biotech. Histochem. 67:363-366. [0009] 3. Jones, R. T. et al. (1993) "Comparison of deparaffinization agents for an automated immunostainer" J. Histotechnology 16:367-369. [0010] 4. Mullin, L. S. et al. (1990) "Toxicology update isoparaffinic hydrocarbons: a summary of physical properties, toxicity studies and human exposure data" J. Appl. Toxicol. 10:135-142. SUMMARY OF THE INVENTION [0011] Compositions and methods are provided for dewaxing wax-embedded biological specimens prior to histochemical or other analyses. The dewaxing compositions and methods provided can effectively remove wax or modified wax-based embedding materials, particularly paraffin or paraffin-based, from specimens prior to histochemical or other analyses, while minimizing danger to users, allowing compatibility with automated use, and maintaining compatibility with downstream analyses. Dewaxing compositions and methods that entail no or limited toxicity or carcinogenicity, produce no or minimal toxic odors, reduce the quantity of toxic solvents used, minimize hazardous wastes, and/or decrease corrosiveness and flammability are provided. The compositions and methods are especially useful for eliminating the use of xylene and for reducing the use of alcohol in preparation of tissue sections for immunohistochemical staining, particularly in hospital laboratories. Dewaxing compositions of the invention comprise a paraffin-solubilizing organic solvent, a polar organic solvent, and a surfactant as specified below in detail. Compositions can optionally comprise water. [0012] A method for dewaxing biological specimens prior to histochemical or other analyses is provided. The method involves contacting a wax-embedded specimen with a dewaxing composition of the invention to solubilize the wax impregnating the specimen prior to histochemical analysis, such as immunohistochemical staining. [0013] It is an object of the invention to eliminate the need for alcohol and alcohol baths for post-dewaxing washes by providing a method which involves contacting a dewaxed specimen, which has been dewaxed by a dewaxing composition of the invention, with an aqueous wash solution comprising a detergent to remove residual dewaxing composition. [0014] A kit for dewaxing a wax-embedded specimen is provided. The kit comprises a dewaxing composition of the invention and can further comprise a second composition of (1) an immunostaining reagent or (2) an aqueous wash solution comprising a detergent for removing residual dewaxing solution. [0015] The present invention eliminates or minimizes the use of toxic organic solvents in immunohistological laboratories. The compositions and methodology described herein effectively remove paraffin or other wax residue from tissue sections and have no adverse effect on the quality of tissue sections prepared for immunohistochemistry. Application of this dewaxing methodology can be extended to other applications where removal of wax from tissue sections are desired, such as in situ hybridization. DESCRIPTION OF SPECIFIC EMBODIMENTS [0016] The present invention provides new dewaxing solvent compositions for removal of paraffin or other waxes from wax-embedded biological specimens for histochemical or other analyses. The compositions comprise a paraffin-solubilizing organic solvent, a polar organic solvent, and a surfactant. In further embodiments the compositions of the invention may be optionally diluted with water. [0017] By "wax" is meant a composition used in the histochemical art for embedding biological specimens for histochemical or other analyses that is solid at room temperature, usually consists of a complex mixture of higher hydrocarbons often including esters of higher fatty acids and higher glycols, may be mineral, natural or synthetic in origin, is harder and more brittle than fats, is soluble in oils and fats, and can optionally contain additives that enhance its specimen-embedding properties. Paraffin is an example of a mineral wax most commonly used in the histochemical field. Paraffin is typically prepared by distillation of petroleum, and is a mixture of primarily solid saturated hydrocarbons. [0018] By "histochemical" is meant to include the techniques and methods known as immunohistochemical, cytochemical, histopathlogic, enzyme histochemical, special stains, microtechnique, in situ hybridization, and the use of molecular probes. Texts illustrating histochemical techniques include "Histochemical and Immunochemical Techniques: Application to pharmacology and toxicology," (1991) Bach, P. and Baker, J., eds., Chapman & Hall, New York, N.Y. pp 1-9, and in "Stains and Cytochemical Methods," (1993) M. A. Hayat, ed., Plenum Press, New York, N.Y., which are incorporated herein by reference. [0019] The paraffin-solubilizing organic solvent is a non-polar hydrocarbon or mixture of hydrocarbons (e.g. as from a petroleum distillate) that has a boiling point well above room temperature, preferably above 110.degree. C., more preferably from about 140.degree. C. to about 250.degree. C., that is in liquid phase at the temperatures used with the present invention (usually 5.degree. to 50.degree. C.), and that is capable of dissolving paraffin used for embedding biological specimens. The paraffin-solubilizing solvent can be a complex mixture of long-chain linear and branched alkane hydrocarbons containing for example esters of fatty acids and higher glycols. The paraffin solubility of the solvent at 25.degree. C. is typically at least 0.1 gram paraffin per 1 liter of solvent, preferably 0.1 gram per 100 ml of solvent, more preferably 0.1 gram per 10 ml of solvent, and most preferably capable of a dissolving an amount of paraffin equal to about 50% of the solvent solutions weight. The paraffin-solubilizing solvent is further miscible with a polar organic solvent when used in a composition of the invention. Examples of paraffin-solubilizing organic solvents include aromatic hydrocarbons, aliphatic hydrocarbons, terpenes, other oils, and petroleum distillates. Preferred paraffin-solubilizing organic solvents have little or no toxic effects. Furthermore preferred solvents are those not classified by the Environmental Protection Agency as hazardous waste. A preferred paraffin-solubilizing solvent furthermore has a flash point higher than about 60.degree. C. which minimizes flammability. A preferred solvent furthermore lacks toxicity, carcinogenicity, and corrosiveness. An isoparaffinic hydrocarbon is an example of a preferred paraffin-solubilizing solvent, in part because of its lack of toxicity, carcinogenicity, corrosiveness and flammability (Mullin et al. 1990). Preferred isoparaffins are branched aliphatic hydrocarbons with a carbon skeleton length ranging from approximately C10 to C15, or mixtures thereof. One preferred isoparaffin hydrocarbon mixture has a flashpoint of about 74.degree. C. Another preferred paraffin-solubilizing solvent is a mixture of C.sub.10 to C.sub.50 branched or linear hydrocarbon chains having a distillation range from a boiling point of 150.degree. C. to about 250.degree. C., and has the general formula of C.sub.n H.sub.(2n.+-.m) where n=10-50 and m=0-4. Mineral spirits is another preferred paraffin-solubilizing organic solvent. A preferred terpene is limonene. Other terpenes that can be used include terpins, terpinenes and terpineols. Less preferably the solvent is an aromatic hydrocarbon solvent such as an alkylbenzene, e.g. xylene, or dialkylbenzene, e.g. toluene. Toluene and xylene are less preferred because of their toxicity and rating as hazardous waste. Furthermore, as discussed below, even when xylene or toluene are used in embodiments of the invention, subsequent alcohol washes are eliminated and replaced with a non-hazardous aqueous wash solution. [0020] The paraffin-solubilizing organic solvent in the composition is typically from about 25% to about 75% by volume of the dewaxing composition. Below the lower percent limit of paraffin-solubilizing organic solvent the dewaxing capability of the composition is significantly decreased. Above the upper limit of paraffin-solubilizing solvent an adverse affect on detergent solubility or water solubility occurs, which adversely affects the effectiveness of a subsequent aqueous wash. The upper limit of solvent can be selected among the upper limit values of 50%, 70%, and 75%, while the lower limit of solvent can be selected from the lower limit values of 25%, 35% and 40%, to obtain a variety of ranges for embodiments of the invention. [0021] Because these compositions are typically prepared by combining components without a precise determination of the final volume of the composition or accounting for volume changes upon mixing, the percentages for each component are qualified with the term "about," with the understanding that one skilled in the art would appreciate the imprecision of the values as a consequence of composition preparation; however, preferably percentage values are taken to mean their precise value when volume changes upon mixing are accounted for. [0022] The polar organic solvent serves the purpose of dissolving the paraffin-solubilizing solvent, surfactant and optionally water. The polar organic solvent is soluble in water to the extent of at least 1 g per 100 g water, preferably 5 g per 100 g water, more preferably 10 g per 100 g water and most preferably the polar organic solvent is miscible with water. Polar organic solvents include ketones and lower alcohols, which include polyhydroxy alcohols and glycols, and lower ethers. Preferred alcohols are C.sub.1 to C.sub.5 alcohols. Most preferred are ethanol, ethylene glycol, isopropanol, propylene glycol and mixtures thereof. A preferred ketone solvent is typically C.sub.3 to C.sub.5 ketone. Most preferred ketone solvents are acetone and methyl ethyl ketone. Preferred ethers are C.sub.2 to C.sub.6 ethers. Particularly preferred polar organic solvents are selected from the group consisting of methanol, ethanol, isopropanol, butanol, tert-butanol, allyl alcohol, acetone, ethylene glycol and propylene glycol, and a mixture thereof. Acetonitrile and dimethylformamide are less preferred polar organic solvents. Furthermore, the polar organic solvent can be a mixture of polar organic solvents. Continue reading about Deparaffinization compositions for dewaxing tissue specimens... Full patent description for Deparaffinization compositions for dewaxing tissue specimens Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Deparaffinization compositions for dewaxing tissue specimens patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. 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