Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving fixed or stabilized – nonliving microorganism,...
Reexamination Certificate
2000-05-16
2002-09-17
Gitomer, Ralph (Department: 1627)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving fixed or stabilized, nonliving microorganism,...
C435S040500
Reexamination Certificate
active
06451551
ABSTRACT:
TECHNICAL FIELD
The present invention relates to methods and compositions for releasing embedded tissue specimens from the embedding medium.
BACKGROUND OF THE INVENTION
Paraffin has been used for many years as an embedding medium in techniques for the preparation of tissue specimens for sectioning in a microtome to produce specimen sections for histological studies. Such embedding techniques 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 (commonly termed “deparaffinizing”), rehydration of tissue sections and staining the sections prior to analysis.
The primary purpose of the embedding medium is to permit the specimens to be sectioned and mounted in an approximation of the natural state. Plastic resins have also been used as embedding medium to provide a harder specimen that allows the 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, 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).
Recent improvements in paraffin-embedding compositions have broadened the applicability of the technique 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) is reported to allow shorter infiltration time and thinner sections (U.S. Pat. No. 4,497,792). Another improvement, the double-embedding technique, yields sections of tissue membranes that usually measure only 10 microns in thickness. In this method, several membranes are fixed and mounted on 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 (trade name 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, R. et al. “A double-embedding technique for thin tissue membranes”
Biotech. Histochem
. 67:363-366 (1992)). Consequently, deparaffmization of fixed, e.g. formalin fixed, paraffin-embedded tissue sections is still a widely used methodology, particularly in hospital histopathology laboratories for immunodiagnostic purposes.
Xylene, which is a flammable, volatile and toxic organic solvent, is 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 treated 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 technique with less toxic and less volatile solvents (Mullin, L. S. et al. “Toxicology update isoparaffmic hydrocarbons: A summary of physical properties, toxicity studies and human exposure data”
J. Appl. Toxicol
. 10:135-142 (1990)). Terpene oil (e.g. available under the trade name AmeriClear from Baxter Health Care Diagnostics, Inc., McGaw Park, Ill.) and isoparaffinic hydrocarbons (e.g. available under the trade name Micro-Clear from Micron Diagnostics, Inc., Fairfax, Va.) produced equal deparaffinization compared to xylene (Jones, R. T. et al. “Comparison of deparaffinization agents for an automated immunostainer”
J. Histotechnology
16:367-369 (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 automated immunostaining devices, is increasing. In these applications, the complexity of the multiple manipulations necessitated by conventional deparaffinization methodology creates a substantial obstacle to the efficient, cost-effective and reproducible handling of embedded tissue specimens.
Accordingly, there remains a need for compositions and methods that can effectively remove, or otherwise eliminate, paraffin, improved paraffin-based and other 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. 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 inflammability are desirable. One such composition and method which has found use is disclosed in PCT Publication WO95/24498, published on Sep. 14, 1995. However, it remains desirable to minimize the use of organic solvents, even those having minimal toxicity or carcinogenicity, odors, hazardous waste concerns, corrosiveness and inflammability.
DISCLOSURE OF THE INVENTION
The present invention provides methods and compositions for releasing the embedding medium from embedded histochemically reactive tissue specimens prior to histochemical or other analyses. In one aspect, the invention provides a method comprising contacting the embedded tissue specimen with a releasing composition comprising a non-polar organic solvent, a polar organic solvent, a surfactant, and water, under conditions sufficient to release a sufficient portion of the embedding medium associated with the histochemically reactive tissue specimen to permit analysis without substantial adverse effect on the histochemical reactivity of the specimen.
The methods provided can effectively remove or otherwise eliminate embedding media, and particularly wax or modified wax-based embedding media, more particularly paraffin or paraffin-based media, from tissue specimens prior to histochemical or other analyses, while minimizing danger to users, allowing compatibility with automated use, and maintaining compatibility with downstream analyses. In this regard, it is considered important to release a portion of the embedding medium associated with the tissue specimen without substantial adverse effect on the histochemical reactivity of the specimen.
The present methods entail no known toxicity or carcinogenicity, no noxious or toxic odors, reduce the quantity of toxic solvents used, minimize hazardous wastes, and/or decrease corrosiveness and inflammability. The methods are especially useful for eliminating the use of xylene and for reducing the use of alcohol in preparation of tissue sections for histochemical staining, particularly in hospital laboratories. Compositions and kits for releasing the embedding medium from an embedded specimen are also provided. The composition comprises a non-polar organic solvent, a polar organic solvent, a surfactant, and water, and the kit comprises a releasing composition of the invention and a second composition of (1) a histochemical staining reagent or (2) an aqueous wash solution for removing, or otherwise eliminating, residual releasing solution.
Other aspects of the present invention will be readily apparent from the following more detailed description.
DETAILED DESCRIPTION OF THE
Chen Taiying
Kalra Krishan L.
Su Sheng-Hui
Zhan Guangrong
BioGenex Laboratories
Gitomer Ralph
The Law Offices of James C. Weseman
Weseman, Esq. James C.
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