Chemistry: analytical and immunological testing – Including sample preparation
Reexamination Certificate
1998-09-16
2002-04-16
Alexander, Lyle A. (Department: 1743)
Chemistry: analytical and immunological testing
Including sample preparation
C436S172000, C435S040500, C435S040520
Reexamination Certificate
active
06372512
ABSTRACT:
BACKGROUND OF THE INVENTION
In present day practice the preparation of organic tissue samples and other material for transmission microscopy, both visible light and electron microscopy, is normally carried out by subjecting the sample to a series of chemical treatments culminating in the production of a solid block in which the sample is embedded. This operation is normally carried out automatically in commercial tissue processors, which are complicated mechanical devices with elaborate temperature control and fluid delivery systems. Their complexity makes them expensive and demanding of frequent technical attention and maintenance. Furthermore, standard tissue processing devices are large and stationary, not portable.
In the conventional tissue preparation process, the tissue is first chemically fixed with formalin, glutaraldehyde, or other materials which serve to preserve the sample from autolysis (self-degradation), and to render the sample rigid as well as to increase its permeability, thereby enhancing the infiltration of subsequent solutions. The infiltration steps which follow are designed to remove all of the water from the sample through progressive replacement with increasing concentrations of solvents such as alcohol and xylene. In the case of conventional light microscopy, infiltration is followed by treatment with melted paraffin, and then the sample is cooled to room temperature whereupon it solidifies. The hardened, infiltrated tissue is then positioned in a mold and surrounded by additional paraffin to produce a tissue block. Microwave ovens have been employed in histology laboratories to speed the penetration of various substances.
In addition to paraffin, plastic is widely employed for producing tissue blocks. The plastic may be methacrylate, epoxy polymer, or related material, but in any case the tissue is subjected to a process roughly similar to that employed in paraffin processing. The water in the tissue is replaced by organic solvents, which are then replaced by liquid polymer. In the case of methacrylate and other water soluble polymers, the dehydration process may be greatly shortened or eliminated. Often the infiltration of the sample by plastic takes place in a small container which also serves as the embedding mold with an excess of polymer that both infiltrates and surrounds the sample, thus infiltration and embedment are combined. The mixed precursor components of the polymer are made to solidify (polymerize) by various means, including exposure to ultraviolet light, heat, or by addition of a chemical catalyst. Plastic processing has been adopted for both light and electron microscopy.
Alternatively, in the frozen section method, fresh unfixed tissue is immediately embedded in a clear, water-soluble, semisolid medium such as O.C.T. compound (Miles, Inc., Elkhart, Ill.) and quickly frozen. This technique is performed to preserve certain tissue components that may be sensitive to extremes of temperature and chemical environment (e.g. antigens) or in the case where microscopic examination of tissue must be accomplished rapidly, such as during some types of cancer surgery. Unlike the “permanent” techniques discussed above, in the frozen section method water is not first extracted from the tissue but instead remains to be frozen along with the encasing embedding medium to form a solid block. The timing of this operation leaves little opportunity for the infiltration/embedding medium to infiltrate the tissue, and so normally the medium serves purely a supportive function.
In conventional optical and electron microscopy, after the block is produced, thin sections of the sample with the surrounding embedding material are cut from the block and transferred to glass slides or other support. The tissue section is then stained with any of a variety of colored or fluorescent dyes for examination. In some techniques the embedding material is chemically removed prior to staining.
Alternatively, methods have been introduced for en bloc staining, wherein the entire sample is stained by immersion prior to being subjected to infiltration and embedment. Sections are then cut from the block for transmission microscopy, or the cut face of the block itself is imaged.
While in some cases the infiltration and embedment steps have been combined, the staining of the tissue has always been implemented separately.
SUMMARY OF THE INVENTION
In general, the invention features a method for preparing an organic sample, e.g., a biological tissue sample, for cutting and subsequent examination; the method involves combining histologic staining, infiltration, and embedding into a single step. Thus, according to the invention, the sample is immersed in a composition composed of: (a) an infiltrating substance; (b) an embedding substance, which can be the same or different from the infiltrating substance; and (c) a stain that chemically associates with the organic sample, and which exhibits different detectable properties when associated and when not associated with the sample. Prior to immersion in the composition, the sample, if it is not a frozen section, preferably is treated with a chemical fixative.
The process preferably employs environmentally sensitive fluorescent stains or dyes that express a specified color or colors (i.e., wavelength) upon being chemically bound to or associated with the sample, but do not express the same specified color or colors when mixed with the embedding medium in the absence of the sample, or express no color when not associated with the sample. This makes it possible to clearly distinguish the tissue or other sample material embedded in the block from the embedding material itself.
As used herein, the term “chemical fixative” refers to a chemical solution that preserves, permeabilizes and aids in hardening the sample. “Infiltrating substance” refers to a liquid or series of liquids that penetrate throughout the tissue to the molecular level and are then transformed into a solid in order to render the sample rigid. “Embedding” or “embedment” refers to positioning the infiltrated tissue in a mold and surrounding it with a substance (usually the same as the infiltrating substance) which is then hardened to form an encasing block. The embedding substance thus serves to provide rigid support and to facilitate the cutting process. “Sectioning” refers to cutting from the block thin slices which may then be mounted on glass slides or other support. “Staining” refers to treating tissue with a colored (including flourescent) substance that associates with the tissue on the molecular level.
Use of conventional, non-environmentally sensitive stains or dyes admixed with the infiltration and embedding medium would cause the embedding medium to express a color or colors similar to that expressed by the tissue, resulting in a loss of contrast between the sample and the infiltration/embedding medium.
The combination of the stain with the infiltration/embedding material reduces the number of steps required to produce embedded tissue for microscopic examination. The invention thus makes possible the production of less complex and expensive automated tissue processing devices. The invention also makes possible, for the first time, a truly portable histopathology laboratory. For example, a compact, field-rated instrument such as is made possible by the invention would find widespread use in military field hospitals, environmental science studies, epidemiology projects, and other special environments such as spacecraft. In civilian medicine, such a device can be incorporated into small satellite laboratories, such as those presently operating within physicians' offices. In this setting they would reduce the expense and turnaround time for the delivery of clinical diagnoses and make a major impact on the overall cost of medicine.
When combined with an automated block sectioning and image acquisition system such as the Surface Imaging Microscope (U.S. Pat. No. 4,960,330, hereby incorporated by reference), the resultant comprehensive system will accept a biopsy as de
Alexander Lyle A.
Clark & Elbing LLP
Resolution Sciences Corporation
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