EXAMINATION

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates generally to the fields of histology and pathology. More specifically, the present invention relates to a novel method of optimizing tissue for pathological examination.

Description of the Related Art

Very thin sections of animal and plant tissue are prepared for many different kinds of microscopic studies by sectioning with a microtome. While the tissue may be cut fresh, the soft and compliant nature of most fresh tissue makes the cutting of undistorted thin sections very difficult. Often, the tissue is cut on a freezing microtome or in a cryostat at temperatures below 0°C, the hardness of the frozen water within the tissue allowing sections as thin as a few micrometers to be cut relatively easily. As these frozen sections are brittle and friable, they are difficult to handle and process further. To simplify sectioning of tissue, a number of procedures have been developed which produce high quality, relatively easy to handle tissue sections. Such procedures typically involve: (1) fixation of the tissue in a solution which insolubilizes and hardens the natural polymers of which tissue cells are composed; (2) dehydration of the tissue through a series of water-miscible, e.g., an alcohol and then paraffin or plastic-monomer- miscible, e.g., toluene or xylene solvents; (3) infiltration of the tissue by melted paraffin or monomer solution; and (4) embedding by freezing the paraffin or polymerizing the monomer to form a solid polymer. See, for example, Staining Methods. J.F.A. McManus and R.W. Mowry (P.B. Hoeber, Inc., N.Y. 1960).

However, there are occassional specimens which remain difficult to section. As the section is cut from the tissue, parts of the cut section tend to fragment and fall from the cut section, or fall from the section as it is removed from the microtome. Several references in the prior art describe procedures for facilitating the cutting and handling of difficult to cut sections. Palmgren (Nature. 174:46, 1954) described the use of a pressure sensitive adhesive tape as a sectioning aid for the

cutting of very large, hard or brittle specimens. A piece of adhesive tape is applied to the surface of a specimen, either frozen or embedded in paraffin block, supported in a microtome. Thus, the section, when cut, is supported by the applied tape. The quality of the uncompressed section of hard, brittle and friable tissue thus produced can be far superior to that of a conventional section of the same block of tissue. However, following the method of Palmgren and processing such a section while it remains on the tape or transferring it to a glass slide involved elaborate, time consuming and inconvenient methods which can also damage the section.

Beckel (Nature. 184:1584, 1959) described the use of Scotch brand No. 810 cellulose-acetate-backed adhesive tape in the process described by Palmgren above. The tape-mounted sections were applied, section side down on wet conventionally albuminized glass slides. After thorough drying, which requires at least a few hours, the adhesive backing, the adhesive layer and the paraffin were all dissolved in tetrahydrofuran for 30 minutes, leaving only the section adhering to the glass slide and available for further processing by conventional techniques. Alternatively, chloroform for 2 minutes, followed by xylene for 30 minutes, can be used for dissolving the adhesive backing, the adhesive layer and the paraffin. Beckel also described a "more rapid method" which used a film of albumin and a solution of 2% celloidin in methyl benzoate or ethyl alcohol to "cement" the section to the glass slide, followed by 1 minute in chloroform and 10 minutes in xylene to complete the treatment.

Gowers and Miller (Nature. 190:425, 1961) attempted to repeat Beckel's method but found that, with available Scotch brand No. 810, the adhesive could not be dissolved, and with the best alternate available tape, Tuck brand No. 200, safe removal of the tape without damaging the section in solvent took from 1 to 10 hours.

Wedeen and Jerno (Am. J. Phvsiol.. 214:776, 1968) used cyanoacrylate (Eastman 910 "superglue") to attach adhesive-tape- supported frozen sections to radiographic (photographic) plates. The cyanoacrylate is initially liquid, but polymerizes to a solid when pressed into a thin film. As the cyanoacrylate polymer is soluble in xylene and other processing solvents which would cause the section to float free, it is not useful for conventional staining procedures.

U.S. Patent No. 4, 545, 831 to Ornstein disclosed a method of positively attaching a tissue section to a microscope slide by a pressure

sensitive polymerizable layer, the curable components of the layer prior to and during polymerization penetrating only very minimally into the specimen and, when polymerized, having a compatible index of refraction and neither affecting nor being affected by and subsequent chemical or physical treatment of the supported tiusse section.

Histopathological analysis of tumorous versus non-tumorous tissue is an art having many of the difficulties described above. When a tumor is excised through conventional methods, it is typically removed as an elliptical specimen of tissue. This tissue is then processed with vertical sections made through the entire length of the block of tissue, analogous to cutting a loaf of bread. Representative sections are then examined under the microscope, and if there is no demonstration of cancer near the lateral or deep edges, the tumor is assumed to be clear in the examined sections. However, these "broad loaf" sections are millimeters in thickness, and, in fact, the tumor may extend to the edges and beyond in sections that are not examined, i.e., tumor remains in the tissue and therefore, the patient.

In contrast to conventional methods of excision and tissue processing, Mohs micrographic surgery is a technique which combines a specialized surgical method with frozen section histopathologic examination in order to optimize cure rates. The Mohs technique attempts to get a full 360 degree view, relative to the center of the tumor, of all of the surrounding edges as well as deep tissue undeneath the tumor. This is done by removing all of the clinically obvious tumor, a process referred to as "debulking" the tumor. Next, the surgeon cuts out a thin layer of what is felt to be normal tissue around and undeneath the defect left following the debulking, creating in essence a hollow bowl shaped piece of tissue. An analogy would be the rim of the bowl representing all of the surrounding skin edges and its base is the piece of tissue taken undeneath the tumor. The exterior most surface of this bowl is most relevant, since if tumor is absent on this surface, then none of the roots of the cancer (which grows without discontinuities) remain on the patient.

A challenge is to convert this three dimensional bowl into a two dimensional surface, which can be examined as a tissue section under the microscope (somewhat analogous to trying to take half an apple that has had its core removed and flatten it so that the peel lies totally flat). To achieve this, in all but the smallest tumors, the tissue is divided into smaller pieces called quadrants (i.e., by bisecting or

quadrisecting the tissue, analogous to dividing the half-apple into quarter apples or smaller sections). Other conventional methods for flattening the tissue include scoring the outer edges of the quadrants, (analogous to slicing the outer edges of a steak to flatten it on a grill), as well as trimming the interior surfaces (analogous to flattening an apple peel by first removing the inner "meat" of the apple).

Following the above flattening techniques, these individual quadrant pieces are then flattened onto a surface on which the tissue block is to be made in preparation for adding embedding media and freezing. This flattening is generally achieved by exerting pressure through pushing on the tissue with "sticks", pressing on the tissue with a glass slide or comparable flat surface, or using flat surfaced tongs (the Miami special) while the tissue is being prepared in a block with embedding media. Subsequently, or while this flattening is occurring, the tissue in its embedding media is cooled to the point of freezing (generally -20 to -40 degrees Celsius), essentially forming an "ice cube". The optimal tissue sections would be obtained if the exterior part of the tissue, representing the deepest and most lateral portions of the tissue, was totally effaced on the surface of the "ice cube" (i.e., the entire surface of the apple peel was flat against one face of the "ice cube").

Very recently, attention has been placed on the optimal way of creating the "ice cube". The "glass slide" technique represents the state of the art method to maximize this flattening. This involves placing the tissue with the relevant, exterior surface face down onto a glass slide and pressing this tissue onto the glass surface with a "stick" or comparable tool. This pressing continues while the embedding media is being applied and the tissue frozen. One studies the undersurface of the transparent slide, and if the tissue "pulls away" from the slide at any focus, the ice block is thawed and the given area is once again pressed into place against the slide. However, the localized pressure and the weight of the embedding media are essentially the only forces causing the curved tissue to become flattened.

The prior art is deficient in the lack of effective means of optimizing tissues for histopathological analysis. The present invention fulfills this longstanding need and desire in the art.

SUMMARY OF THE INVENTION

In one embodiment of the present invention, there is provided a method of optimizing a thin tissue section for histopathological analysis, comprising the steps of: contacting said tissue section with a microscope slide, said slide bearing on at least one surface a temperature sensitive adhesive compound.

In another embodiment of the present invention, there is provided a method for detaching the tissue from the device of the present invention in preparation for histologic examination, comprising the step of disengaging said tissue from said adhesive by exerting a mechanical forces against said tissue.

In yet another embodiment of the present invention, there is provided a device for preparing a tissue block with tissue optimally effaced on one face and of a given height and wherein the device is a mold for embedding the tissue or preparing the tissue block which attaches to the device of the present invention.

In still yet another embodiment of the present invention, there is provided a method of cutting tissue from a block of tissue comprising the step of pressing a glass slide against the surface of the tissue or a tissue block while the tissue is being cut into thin sections.

In still yet another embodiment of the present invention, there is provided a method of optimizing gross tissue for pathological analysis, comprising the steps of: contacting said tissue with a device having a rigid, flat first element, said first element bearing on one surface a first temperature sensitive adhesive component; said first component adhered to a flexible second element, said second element bearing a second temperature sensitive adhesive component on its surface; and performing a pathological analysis. Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention given for the purpose of disclosure.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a method of optimizing a thin tissue section for pathological analysis, comprising the steps of: contacting said tissue section with a rigid, flat device, said device

bearing on at least one surface a temperature sensitive adhesive compound. Preferably, the temperature sensitive adhesive compound should adhere to tissue at temperatures of from about 25°C to about 200°C and should lose its adhesive properties in temperatures from about 20°C to about -40°C.

In one respect, the temperature sensitive adhesive compound retards the curling of the tissue. In another respect, the temperature sensitive adhesive compound attaches to a tissue section and improves the flattening of the tissue section. Preferably, the adhesive component is resistant to degradation at a temperature of from about 25°C to about 200°C.

Generally, the temperature sensitive adhesive compound is selected from the group consisting of Scotch tape, cellulose-acetate- backed adhesive tape, Scotch brand No.810, Tuck brand No. 200, cyanoacrylate and Eastman 910. Preferably, the adhesive is relatively insoluble in formalin or other fixative solutions enabling the tissue section to become fixed while it is attached to said device. For example, it is desirable that the adhesive is resistant to degradation at a temperature used in microwave processing of tissues in preparation for histologic examination.

The present invention also provides a novel device used in the method described above. In the most preferred embodiment, the device is a microscope slide. Generally, the device is used in preparing tissue for fixation and/or embedding into a tissue block. That is, the device is useful in the preparation for thin sectioning of the tissue for histopathologic examination. For example, the device may be used to orient and position tissue during formalin fixing and/or paraffin embedding, frozen section analysis or microwave processing. The present invention also provides a method for detaching the tissue from the device of the present invention in preparation for pathologic examination, comprising the step of disengaging said tissue from said adhesive by exerting a mechanical forces against said tissue. In one embodiment, the tissue is sliced away from the device using a cutting instrument. The present invention also provides a method of detaching a fixed or embedded tissue from the device of claim 1 in preparation for pathologic examination comprising the step of mechanically disengaging the tissue from said device. In one embodiment, the temperature is

changed so that the adhesive properties of said component is significantly reduced so as to facilitate detachment.

The present invention also provides a device for preparing a tissue block with tissue optimally effaced on one face and of a given height and wherein the device is a mold for embedding the tissue or preparing the tissue block which attaches to the device described above. Preferably, the mold enables the embedded tissue or tissue block to have a precise height and a fixed angle on the surface opposite to the effaced tissue. In one embodiment, the device has a screw on the opposite side of the effaced tissue, said screw allowing a precise height to be achieved to the block, while providing a means of advancing said tissue block. In addition, the device may have channels or vents in the device enabling the level of the embedding fluid to be assessed and allowing for venting. Current art includes making the mold in a block but does not provide the same precision of the height nor advancement features of the present invention because the tissue block is merely "popped out" of the bolt and then transferred to another device for advancement during cutting.

The present invention also provides a method of optimizing gross tissue for histopathological analysis, comprising the steps of: contacting said tissue with a device having a rigid, flat first element, said first element bearing on one surface a first temperature sensitive adhesive component; said first component adhered to a flexible second element, said second element bearing a second temperature sensitive adhesive component on its surface; and performing a histopathological analysis. Preferably, the temperature sensitive adhesive components adhere to tissue at temperatures of from about 25°C to about 200°C and said component loses its adhesive properties in temperatures from about 20°C to about -40°C. Preferably, e element is selected from the group consisting of Scotch tape, cellulose-acetate-backed adhesive tape, Scotch brand No. 810, Tuck brand No. 200, cyanoacrylate and Eastman 910.

It is an object of the present invention to use precoated slides having a flat surface in order to maximize the efficiency of achieving the optimal slide for achieving margin control of a tissue block. It is important to create a relevant flat and effaced tissue on the block as done by the adhesive used herein and to have the tissue embedded in the block opposite the surface at a fixed angle relative to the tissue surface, e.g., parallel faces. This characteristic would enable

the tissue to be more readily advanced for sectioning. It is further important to maximize the efficiency of producing an initial section of tissue that represents a relevant margin that can then be examined histopathologically.

The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion.

EXAMPLE 1

A New Method of Cutting Tissue Sections

Tissue that is prepared for frozen section pathology involves making a "ice cube" block of tissue and then taking parallel thin seotions, beginning at one face and cutting deeper for histopathologic examinations. Currently, microtomes are used where the block of tissue advances forward while being raised and lowered towards a blade which is in a fixed position. Thus, 2 to 20 micron sections are achieved by raising the tissue block above the blade, advancing this block 2 to 20 microns, and lowering the block onto the blade so that the thin section peels off of the blade, and repeating this procedure to obtain deeper sections into the block.

Two technical difficulties accompany this procedure. First, while the blade's cutting surface is extremely sharp and narrow, the width of the remainder of the blade precludes having the glass slide to which this section is to be mounted, from coming in direct contact with the tissue section as it is being cut. The transfer of the tissue off of the blade onto the glass slide is, in fact, one of the most technically demanding aspects of performing thin sectioning of tissue. Secondly, as a related issue, the tissue that peals away has very little structural integrity, due to its thinness, and often fragments or folds over itself making histologic interpretation difficult, if not impossible. To compensate, one often has to cut deeply into the block in order to achieve acceptable sections. Consequently, critical information based on the first, deepest sections of the tissue blocks is often lost, serving as a significant limitation, particularly if orientation has been optimized by the methodology described herein.

FY AMPLF. 2

Preparation of Tissue Blocks

Following the flattening of the tissue for Mohs sections, using the techniques described above, an "ice cube" of tissue and embedding media is formed. As detailed previously, the key issue is to have the tissue flat and effaced on the front side of the "ice cube", which is more aptly described as an ice cylinder. Once this goal is achieved, a second objective then naturally evolves of having the back side parallel or at a fixed angle relative to the front (tissue) side, and the height of the cylinder being relatively fixed. The first objective would aid in having the block of tissue advance in the microtome in such a way that the angle that the block of tissue presents itself to the microtome blade is fixed and is parallel or at a fixed angle relative to the blade, and therefore does not have to be adjusted from block to block. Furthermore, this orientation could be "pretested" with a "ice cube" made of embedding media without tissue to ensure that the orientation is optimally parallel, without sacrificing any of the crucial "exterior" tissue from a section of tissue from a patient. The second goal adds to the convenience of advancing the block a relatively fixed distance each time it is being presented to a cutting surface. This represented a variation of a model described herein, namely a cylindrical mold such as the plastic middle to a roll of tape. This plastic cylinder had its top half cut off, leaving a flat rim on the sides to enable embedding media overflow to spill, while maintaining a flat top surface of media over the central hole where the tissue was laying on a glass slide). The device involves using a screw to "push out" the block of ice after it is frozen inside of a mold consisting of a vented bolt, having holes at a fixed height. Here, the bolt could be attached to the glass slide covered with an adhesive and serve as a mold for the embedding media. Once a level of the media is achieved that a small leak occurs through small horizontally oriented vents, the screw is threaded through the top part of the bolt. This should add to the flattening force on the tissue. To prevent the glass slide from popping off from the bolt or the embedding media leaking from its bottom edges, vents would also be placed on the top of the bolt (oriented at an incline) and/or up the screw itself. Once the embedding fluid reached a level on the(se) vent(s), the glass slide/tissue/bolt/scrow unit would be ready for cooling so that it becomes a frozen block. Once the block was frozen,

the other advantages of the screw would be utilized. These include the mechanical advantage of the screw to have the frozen tissue pushed out from its mold/block, as well as being able to use the thread of the bolt to precisely and accurately have thin sections of tissue pushed out in preparation for cutting, either with a microtome blade, or with the cutting wire described above. This could be done in a variety of ways, but the preferred model involves having the screw connected to a series of reducing gears, which gives a mechanical advantage to turning the screw, as well as enhance the precision of the amount of the "ice cube" block is advanced as the screw is turned.

Research on the materials to make the boltscrew combination, the dimensions and placement of the vents, the optimal thread, and the reducing gears that would be connected to the device and ways in which it could be displaced in association with cutting are routine and could be conducted without undue experimentation by those having ordinary skill in this art.

EXAMPLE 3

The Use of an Adhesive to Optimize Tissue Flattening

The use of an adhesive optimizes tissue flattening by reducing the likelihood of false positive margins. The purpose of an adhesive is to resist the tendency of elastic forces to curl the tissue back to its original state while the tissue is being made into its "ice cube" state. The adhesive used in the methods of the present invention has several characteristics essential to this specific desired function. The adhesive must adhere to tissue at temperatures around room temperatures. This function of the adhesive is vital to ensuring that the tissue does not curl. Merely cooling a glass slide, which makes it slightly sticky to tissue is not sufficiently adherent to maintain the desired flattening of the tissue nor is the use of commercially available slide coatings such as poly-L-lysine. Secondly, the adhesive must lose its adhesive properties in colder temperature, e.g., -20°C to -40°C where frozen sections of skin are typically processed so that the "ice cube" produced by cooling tissue embedded in the mounting media can be easily removed. Alternatively, if the initial tissue placement on the flat surface is not optimal then prior to placing a mold on top and adding mounting media, the tissue/adhesive can be cooled, resulting in less adherence and the tissue peeled off and replaced on a room

temperature adhesive/flat surface unit. Finally, the surface that the adhesive is on should be flat and relatively inflexible to prevent bending and therefore soft plastics are not preferred. However, a flexible second element could be attached to the rigid element, and this flexible element would have an adhesive surface that comes in contact with the tissue. This would have advantages or facilitating detachment of the tissue from the rigid device, in preparation for sectioning, as well as in preparation of the gross tissue, i.e., sectioning, would could be done on this flexible element prior to the final flattening stage. An advantage of a transparent substance such -as a glass slide is that one can look at the tissue from the undersurface and assess whether the relevant deep surface and edges are all suitably positioned prior to adding mounting media and creating the "ice cube", i.e., ensuring there is no "tenting" of the bottom surface or curling of an edge, thereby minimizing false positive and false negative sections. This adhesive technique can also be used to flatten tissue and maintain orientation in preparation for sections using paraffin embedding, i.e., during formalin fixation or other processing techniques such as microwave preparation. Any patents or publications mentioned in this specification are indicative of the levels of those skilled in the art to which the invention pertains. These patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

One skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The present examples along with the methods, procedures, treatments, molecules, and specific compounds described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention as defined by the scope of the claims.