Apparatus and method for preparing frozen tissue specimens

An apparatus and method for preparing frozen tissue specimens includes a base supporting a pair of rotary motion platforms and a center platform. The rotary motion platforms are movable from an open, side-by-side position to a closed, center platform-covering position. The rotary motion platform includes a plurality of cryogenic discs, each identified for receiving a plurality of tissue samples. The center platform includes a plurality of cryogenic discs, each having a plurality of bores for receiving object holders thereon, each object holder for receiving a frozen specimen thereon. The center platform cryogenic discs each have a channel system with intersecting peripheral, chordal and radial channels. A moistening tray includes structure having a closed, object holder wetting position and an open, object holder elevating position. Further disclosed is the use of a tissue orientation map, usable on temporary tattoos and tissue receiving sheets for specimen orientation and mapping of tumor roots. Also disclosed are labels and identification methods, fiber-reinforced embedding compounds and the use of treated polyester sheets for tissue specimen placement.

BACKGROUND OF THE INVENTION

The present invention is broadly directed to an improved apparatus and method for rapidly freezing a plurality of tissue specimens at cryogenic temperatures that enhances heat transfer, quickly cools tissue holders and tissue, facilitates cutting of thin tissue sections and facilitates tracking of tissue specimens throughout a tissue preparation and examination process. More particularly, the invention is directed to a multi-specimen tissue freezing apparatus and method of mapping and labeling, the apparatus including a channel system permitting highly effective circulation of a cryogen and transfer of heat thereto so as to rapidly cool tissue specimens.

Biopsy or surgical removal of tissue specimens for histologic examination is commonly employed for diagnostic purposes. When a lesion is known or suspected to be malignant, the entire mass is generally excised, if possible. An examination technique may be employed in which the entire tumor margin surface area is reviewed under a microscope. This technique involves microscopic screening of the exterior surface area of the tumor for the presence of malignant cells in order to ensure that all such cells have been removed. If practiced effectively, tumor margin surface area examination enhances the likelihood of complete removal of all cells of a localized malignancy.

Once harvested, the tissue sample is preferably quickly frozen at a controlled rate using a cryogenic coolant in order to obtain high quality frozen sections suitable for use in diagnosis. The tissue is then cut into thin layers or sections for histological examination. It is important that the tissue be frozen and the histologic examination performed as quickly as possible, since the patient must be kept waiting pending the microscopic evaluation, in case any additional tissue must be excised. In the past each review of the tissue was comparatively lengthy, so that a patient had to be maintained in a very uncomfortable state with an open wound for a long period of time. Much of the delay was due to slow freezing of the tissue samples, so fast freezing is very desirable, especially where multiple samples must be taken.

Controlled freezing of the tissue may be accomplished using the methods and devices set forth in Applicant's previous patents, such as U.S. Pat. Nos. 4,695,339; 4,752,347; 5,628,197; 5,829,256; 6,094,923, 6,289,682 and 6,725,673, which are incorporated herein by reference. The rate at which specimens can be frozen under such controlled conditions is determined by the rate of heat transfer from a cryogenic fluid, such as liquid nitrogen, to the platform on which the tissue is placed. Specimens must be frozen relatively quickly in order to avoid formation of large ice crystals. However, attempts to increase the rate of freezing by use of excessive amounts of cryogenic material may impair control over the freezing process. Specimens that are frozen unevenly or incorrectly may be marred by voids and artifacts that might impair histologic examination and diagnosis. It is also desirable to minimize the quantity of cryogenic fluid that is used, since such fluids are costly and may present certain environmental hazards which must be addressed. Therefore, it is important to enhance heat transfer while maintaining control over specimen freezing conditions and conserving use of cryogenic fluids.

Even a properly prepared tissue specimen that is quickly frozen under controlled conditions may not result in a high quality histologic specimen unless thin tissue sections can be taken easily from the frozen specimen. Compression of the section may occur where difficulty is encountered in cutting thin sections from a frozen specimen. Upon gross examination, compressed tissue sections may appear to be usable for mounting on slides, but will prove to be difficult to evaluate. Badly crumpled sections may be unusable.

Another problem faced in a busy laboratory is the proper identification of tissue samples as they travel from station to station, through the processes of tissue harvesting; preparations, such as relaxing and anatomic color marking before freezing; freezing which may include more than one step or transfer to and from freezing platforms; slicing; and examination. A further challenge is developing improved methods of mapping or marking both the patient and the tissue sample to ensure correct orientation of the tissue sample with respect to the patient if clinical reorientation is necessary for further tissue harvesting. Accordingly, there is a need for apparatus and methods for evenly and quickly freezing multiple tissue specimens under controlled conditions with correct labeling and mapping.

SUMMARY OF THE INVENTION

An apparatus according to the invention includes a base supporting at least one rotary motion platform and a center platform. The rotary motion platform or platforms are movable from an open, side-by-side position with respect to the center platform to a closed, center platform-covering position. Each platform includes a plurality of cryogenic discs, each identified with numerals printed or otherwise located thereon to aid in the placement and tracking of a plurality of tissue specimens during freezing and transfer of the specimens onto object holders.

Each rotary platform cryodisc is also equipped with a channel system for circulation of a cryogenic fluid within the disc structure that includes a peripheral channel and connecting radial channels. The center platform, that includes a plurality of bores for receiving object holders thereon, includes a channel system with a plurality of chordal and radial channels disposed between the bores, the chordal and radial channels communicating with a peripheral channel. Each of the cryodiscs includes a circumferential ring seal. The discs of the center platform include peripheral inlet and outlet ports.

According to an aspect of the invention, a moistening tray is provided for receiving the object holders and wetting undersides thereof with alcohol prior to placement of the object holders in the bores of the center cryodisc. The moistening tray includes a moistening pad and a holding structure. The holding structure has two positions: a first closed position wherein the object holders are received by the moistening tray with undersides thereof contacting the moistening pad; and a second open position wherein at least a portion of the object holders are in spaced relation with the moistening pad.

A method of quick freezing a tissue specimen by cooling the specimen on a rotary cryogenic disc and then transferring the specimen to an object holder includes the steps of placing up to a plurality of specimens on a single cryodisc to be cooled and then placing a unique label adjacent to each specimen on the cryodisc prior to cooling, each label having an identification sequence embedded throughout a thickness of the label, the label linking the particular specimen with a particular patient then traveling with the specimen throughout the freezing, transfer, slicing and examination steps of the process.

Another aspect of the invention includes the application of an embedding medium to each specimen prior to the transfer of the specimen to a corresponding object holder. Preferably, the embedding medium includes both fiber and an electrically conductive polymer. Additionally, the fiber-reinforced medium may include protein, such as a silk fiber. A preferred fiber for use in the embedding medium is bamboo cellulose. The electrically conductive polymer may be polyaniline or a long chain polyaniline emeraldine salt grafted to lignin.

In another alternative aspect of the invention, tissue specimens are placed on a sheet of surface treated polyester film rather than directly on the cryodisc to be cooled. Preferably, the surface of the polyester film is treated by brushing with albumin.

A further aspect of the invention is a tissue orienting pattern or grid for placement, for example on a patient in the form of a temporary tattoo that corresponds to markings on the tissue specimen. Such a grid or pattern may also be used on the tissue receiving polyester film previously described herein. The tissue orienting pattern aids in the location and ongoing tracking of tumor roots. A temporary tattoo having a tissue orienting pattern thereon may be applied to a patient prior to harvesting a tissue specimen therefrom with a portion of the tattoo remaining on the patient after tissue harvesting. A tissue orienting grid or pattern according to the invention preferably includes an X axis, a Y axis, and concentric circles, the X and Y axes including color coding cooperating with color coding on the tissue specimen.

OBJECTS AND ADVANTAGES OF THE INVENTION

Therefore, objects of the present invention include: providing an apparatus and method for rapidly freezing tissue samples; to provide such apparatus and methods wherein cryogenic fluid is used to rapidly cool discs associated with the receiving of opposite sides of a plurality of tissue samples; to provide such apparatus and methods that aid in the identification and tracking of a plurality of tissue samples; to provide such apparatus and methods to aid in the mapping of tissue samples if further tissue harvesting is deemed necessary; and to provide such apparatus and methods to aid in transfer of the tissue specimens through the various process steps.

DETAILED DESCRIPTION OF THE INVENTION

An apparatus generally indicated by the reference numeral1for preparing frozen tissue specimens in accordance with the present invention is depicted inFIGS. 1-16, and includes a platform mechanism2and a fluid transfer system3. With reference toFIG. 1, the platform mechanism2includes a base4supporting fixed, upstanding front and rear support panels5and6. The panels5and6support between them a pair of laterally spaced, generally horizontal columns7and8in vertically spaced relation to the base4. A central linear motion platform9is located between the support panels5and6and is supported on the base4by well known structure permitting the platform9to be raised and lowered in spaced relation to the base4. The support columns7and8are coupled with respective rotary motion platforms10and11in laterally spaced relation to the central platform9and in vertically spaced relation to the base4. The columns7and8are pivotally coupled with the support panels5and6, permitting selective axial rotation of the columns7and8and the respective rotary motion platform10and11from an open position, in which the platform10or11is laterally adjacent to the central platform9, to a covering position, in which the platform10or11is vertically adjacent the central platform9.

In addition to the respective rotary motion platforms10and11, the columns7and8also support associated components of the fluid transfer system3, best shown partially schematically inFIGS. 6 and 9. The fluid transfer system3includes flexible cryogen supply conduits12, which are coupled with a source (not shown) of a liquid cryogenic material, such as liquid nitrogen, and return conduits13. While the illustrated embodiment is designed for use with liquid nitrogen because it is generally inert and non-combustible, the apparatus1may be used in conjunction with other cryogenic fluids.

With reference toFIGS. 1-6and14, each of the rotary motion platforms10and11includes a series of four spaced cryodiscs16(designated “rotary” cryodiscs, for clarity) for receiving tissue specimens17. Each rotary cryodisc16is also encircled by a groove18that is in fluidic communication with a vacuum pump (not shown). Encircling a periphery of each groove18is a groove19filled with a pressure sensitive adhesive. A sheet of a plastic film20is supplied for placement over one or more specimens17placed on each cryodisc16as shown inFIG. 1in covering relationship to the grooves18and19, and a vacuum is drawn through the groove18. The adhesive-filled grooves19allow for quick and ready placement and attachment of the plastic film20to the platform10or11and further provides a seal between the platform10or11and the film when vacuum is pulled through the grooves18. As shown inFIG. 14, the vacuum then serves to draw the film sheet20tightly against the specimen or specimens17, the cryodisc16and the surface of the rotary motion platform10or11. In this manner, the plastic film20compresses the specimens17against the cryodisc16and air pockets between the specimens17and the cryodisc16are drawn radially outward and removed by the vacuum.

The grooves19are preferably filled with a pressure sensitive adhesive that is first melted and poured into the grooves19. Such an adhesive adheres to the plastic film20but allows for easy removal of the film20. Although vacuum grease may be utilized to provide a seal between the film20and the platforms10or11, a pressure sensitive adhesive is preferred as it is not necessary to replace the adhesive each time the apparatus1is used. A preferred pressure sensitive adhesive is produced by the HB Fuller Company under the product designation HM1478. Alternatively, rubber cement or white rubber may be utilized in the grooves19.

In an alternative embodiment, a single adhesive groove (not shown) extends along a periphery of each of the platforms10and11, surrounding all four cryodiscs16on the platform10or11. Such a groove allows for the placement of one large sheet of plastic film (not shown) over all of the tissue specimens on all four cryodiscs16of a platform10or11, rather than the placement of smaller individual plastic sheets over each cryodisc16.

With particular reference toFIGS. 1 and 2, in order to easily identify tissue specimens and track such specimens during processes of the invention, each cryodisc16is separated into three equal sections. A top surface21of the cryodisc16is divided into three sections by equidistant radially extending lines21a,21band21c. The lines21a,21band21cmay be made by engraving and then filling with a bright colored paint. The three sections formed by such lines have a numeral from one to twelve engraved or otherwise imprinted on both the individual section and on the platform10or11adjacent the particular section to aid in identification when the individual sections are covered with a tissue specimen17.

With reference to FIGS.1and7-11, the linear motion platform9has four cryodiscs22somewhat similar to the rotary cryodiscs16that are sized and spaced to cooperate with the rotary cryodiscs on the rotary motion platforms10and11. The cryodiscs22are designated herein as linear cryodiscs22to distinguish from the rotary cryodiscs16. Formed in each cryodisc22are a central hollow bore23and a plurality of evenly spaced peripheral hollow bores24. In the illustrated embodiment there are three evenly spaced hollow bores24located outwardly radially from the central hollow bore23. Each of the bores23and24are sized for receiving a stem26of a tissue-receiving plate or object holder27, best shown inFIGS. 1,8and9. The bore23provides for the use of a single larger object holder (not shown) accommodating a larger tissue specimen (not shown). In the illustrated embodiment, three object holders27, each accommodating a smaller tissue specimen17may be placed on each cryodisc22.

With particular reference toFIGS. 1 and 7, in order to easily identify tissue specimens and track such during processes of the invention, a top surface28of the four linear motion cryodiscs22and the platform9are marked with the numerals one to twelve, cooperating with placement of such numerals on the platforms10and11, so that a tissue specimen placed, for example, on a section identified with the numeral “5” of the cryodisc16is transferred to an object holder27at an area of a cryodisc22also identified, for example, with the numeral “5”.

Returning to a description of the rotary platform cryodiscs16, illustrated inFIGS. 2-6, each disc16includes the top or upper surface21that is substantially circular, a substantially circular bottom or lower surface31and an annular circumferential sealing ring or seal33extending between the surfaces21and31. Each cryodisc16is equipped with a channel system, generally34, for circulation throughout the cryodisc16of a cryogenic fluid delivered via the fluid transfer system3. The illustrated cryodisc top surface21is generally planar and smooth, for receiving the tissue specimens17. The rotary cryodisc top surface21is preferably coated with a polymeric composition, especially a tetrafluoroethylene, such as is sold under the trademark Teflon® by Du Pont, to facilitate quick release of the specimens17. The bottom surface31is also generally planar and smooth and includes a central stem38that is apertured to provide an inlet port39for coupling with the supply conduit12through a nipple40. The bottom surface31also includes a pair of circumferentially spaced apertures or outlet ports41, for coupling with the return conduits13by means of nipples42. The sealing ring33also includes a top or upper surface43and a bottom or lower surface44with a sidewall45therebetween.

The top and bottom surfaces21and31of the cryodisc16are illustrated inFIGS. 2 and 5as substantially circular in shape and identical in diameter, with the sealing ring33sized to encircle the disc sidewall32in snug or generally sealing relationship. The sealing ring top and bottom surfaces43and44are aligned so as to be contiguous with and extend generally flush with respect to the cryodisc16top and bottom surfaces21and31respectively.

As illustrated inFIGS. 3,4and6, each rotary cryodisc16includes the channel system34that has a circular manifold configuration for circulation of a cryogenic fluid throughout the cryodisc16. The channel system34includes a circumferential groove or perimeter channel50and an axial reservoir area or collection chamber51which is concentric with the inlet port39. A series of spaced radial, but centrally converging, bores or channels52communicate between the circumferential channel50and the reservoir51. The radial channels52are each equipped with a series of spaced and radially inward projecting fins, ridges or serrations53for operably increasing turbulence in the cryogenic fluid and enhancing heat transfer from the cryodisc16to the cryogenic fluid. Two flow-directing dams54are provided to block the flow of cryogenic fluid directly from the reservoir51to the outlet ports41.

The cryodiscs22disposed on the central linear motion platform9that are illustrated inFIGS. 7-11each include a top or upper surface60and a bottom or lower surface61. The bores23and24extend between the top and bottom surfaces60and61. A sealing ring or seal64is configured for mated sealing engagement with the cryodisc22about a circumference thereof, sealing at both the top and bottom surfaces60and61. Each cryodisc22also is equipped with a channel system65somewhat similar to that of the rotary platform cryodiscs16and used for circulation of the same cryogenic material delivered via the fluid transfer system3. The apparatus1may also be equipped with a heating element67disposed beneath the cryodiscs22near the bottom surface61and controlled by a thermostat (not shown), for warming the cryodiscs22to a desired temperature after usage, so as to be ready for a next usage.

The cryodisc22top surface60is generally planar and smooth, for supporting the tissue receiving object holders27and providing maximum thermal contact for heat transfer between the object holders27and the cryodisc22. Peripherally spaced inlet and outlet ports70and71respectively, communicate with the channel system65and couple with respective supply and return conduits12and13via respective nipples73and74. As shown inFIGS. 7 and 9, the sealing ring64is generally L-shaped when viewed in cross-section including a radially outward extending upper flange portion82having upper and lower surfaces83and84, respectively, and a lower, disc-circumscribing portion85having an outer sidewall or skirt portion86, an inner sidewall87, and a lower or bottom surface88therebetween.

The top and bottom surfaces60and61of the linear cryodisc22are generally circular in shape and identical in diameter, with the sealing ring64sized to encircle the disc22in substantially sealing relationship with the sealing ring flange upper surface83aligned to form a contiguous surface with the disc top surface60and the sealing ring lower portion bottom surface88aligned to form a substantially flush, contiguous surface with the disc bottom surface61. This construction permits the top surface60of the cryodisc22to extend radially outwardly beyond the lower portion85of the seal64. In this manner, the mass of the cryodisc22to be cooled is reduced in proportion to the size of the usable surface, thus minimizing the quantity of cryogenic fluid necessary to lower the temperature of the cryodisc22and the tissue specimen17. While the upper flange82and the lower portion85are depicted herein as being of unitary construction, it is foreseen that the flange portion82may be of unitary construction with the top surface60of the linear disc22, with the lower portion85serving as a sealing ring. It is also foreseen that the shape of the top surface60including the flange portion82when viewed from above may be altered to a non-circular configuration, such as for example, triangular or other multilateral, ellipsoid or eccentric shape.

The channel system65illustrated inFIGS. 9 and 11includes a substantially circular, circumferential, perimeter groove or channel90, a plurality of intersecting chordal channels92flow connected to the perimeter channel90at both ends thereof, and radial channels94extending from near the bore23to the perimeter channel90. Specifically, pairs of chordal channels92flow on either side of each of the bores24. A group of three adjacent radial channels94flows outwardly from a central area of each of the chordal channels92. There is also a radial channel94evenly spaced between each of the three bores24, one of which is an inlet channel96directly flow connected to the inlet port70. The intersection of the chordal92and radial94channels near the center bore23creates an axial reservoir area or collection chamber98that surrounds the bore23. The inlet channel96that is directly flow connected to the inlet port70is also directly flow connected to the collection chamber or area98, but not directly connected to the outer or perimeter channel90. The outlet port71is directly flow connected to the outer channel90, but not directly flow connected to the collection chamber or area98. Similar to the channel system34of the rotary cryodisc16, the channels92and94may be equipped with spaced fins, ridges or serrations100for producing turbulence and enhancing heat transfer.

The cryodiscs16and22are both preferably constructed of a material having a high coefficient of heat transfer, such as a metal, with aluminum being particularly preferred. The circumferential sealing rings33and64are preferably constructed of a heat-shrink aluminum alloy to ensure a tight seal between the discs16and22and their respective rings33and64. Any other suitable thermally conductive material may also be employed. The channel systems34and65are preferably constructed by drilling, although it is foreseen that they may also be of cast or molded construction. The fins53and100are formed by threading or tapping, or by other suitable means.

While the outstanding upper flanges82of the sealing rings64for use with the linear platform cryodiscs22advantageously reduce the thermal mass of the cryodiscs22to be cooled, it is foreseen that the rings64may be constructed without the flanges82, with a structure similar to the sealing rings33for use with the rotary cryodiscs16. It is also foreseen that the sealing rings33for use with the rotary cryodiscs16may be constructed to include flange structure similar to the flanges82.

In use, a quantity of liquid nitrogen or other cryogenic fluid is conveyed via the supply conduits12from a storage vessel (not shown) to the inlet ports39and70of the cryodiscs16and22. In the case of the rotary motion platform cryodiscs16, the supply conduit12conveys the liquid nitrogen through the inlet port39and into the axial reservoir51. The nitrogen flows outwardly from the reservoir51, into the radial channels52, where it passes over the fins53. The fins53cause turbidity in the flow, which enhances heat transfer from the structure of the cryodisc16to the liquid nitrogen. Portions of the liquid nitrogen encounter the flow-directing dams54, that prevent the liquid from exiting directly from the outlet ports41. Nitrogen gas flows into the perimeter channel50, which is sealed by the sealing ring33to prevent escape of gas or liquid to the atmosphere. The nitrogen gas travels around the perimeter channel50until it reaches the outlet ports41, where the gas is conveyed away via return conduits13.

A portion of the supply conduit system12also conveys a quantity of liquid nitrogen from the storage vessel (not shown) to each inlet port70of the linear platform cryodiscs22. The fluid travels through the inlet channel96to the central reservoir area98that surrounds the central bore23and outwardly through the chordal and radial channels92and94. As the liquid nitrogen warms and gasifies, nitrogen gas passes outwardly from the perimeter channel90through the outlet port71and is conveyed away via the return conduits13.

A control panel101is mounted on the front of the apparatus1for use by an operator in control and use of the apparatus1. An object holder moistening tray, generally106, may be mounted on or placed near the apparatus1for ready access to the object holders27.

The moistening tray106illustrated inFIGS. 1,12and13holds up to twelve small object holders27(six are shown) that cooperate with the three peripheral bores24formed in each of the linear platform cryodiscs22. Alternatively, the moistening tray106may be used to hold four larger object holders (not shown) that cooperate with the central bore23of each of the linear platform cryodiscs22. Whether the object holder27or a larger object holder (not shown) is utilized, the moistening tray106allows for ready access to object holders moistened with isopropyl alcohol on underside surfaces108thereof.

In the illustrated embodiment, each object holder27includes the previously identified stem26thereof that is substantially cylindrical in shape and a circular plate110having a top surface112and the afore mentioned underside surface108. Formed in the top surface112are circular concentric grooves114that aid in holding a frozen tissue specimen17as will be described in more detail below. The stem26protrudes centrally from the plate110underside surface108and is integral or otherwise attached thereto. The illustrated object holders27have plates110that are approximately40mm in diameter, but may be larger or smaller, depending on the preference of the user and relative size of cooperating equipment. The illustrated embodiment provides for the rapid freezing of up to twelve tissue specimens17during a single freezing cycle of the apparatus1. The object holder stem26is also sized and shaped to cooperate with an apparatus (not shown) for holding the object holder27during slicing of the frozen tissue specimen17in preparation for microscopic examination.

The illustrated moistening tray106is a rectangular container having a box-like body119that includes a base120and side walls122, and further includes a rectangular lid124with a glass window125and a hinge126attaching the lid124to the body119. Within the container body119is an object holder holding and elevating structure, generally130, that includes a platform structure132having spaced bores134formed therein; a plurality of rods136attached to the platform structure132with springs138; a pair of elongate bars140slidable with respect to the platform structure132, each having sloping, curved surfaces142formed therein for operable engagement with the spring-loaded rods136; a pair of levers144cooperating with both the slidable bars140and the lid124; and a moistening pad148saturated with alcohol, preferably 70% isopropyl alcohol, the pad148having spaced apertures150cooperating with the bores134.

The platform structure132fits snugly within the box-like body119. In the illustrated embodiment, the structure132is of substantially solid construction, with the exception of the bores134, two lower substantially horizontal spaced grooves150sized and shaped for receiving the sliding bars140, the grooves partially defined by an end wall or stop151; and grooves(not shown) perpendicular to the grooves150for receiving the rods136and springs138. The bores134and the pad apertures148are each sized to receive the stem26of an object holder27, with the underside108thereof in contact with the moistening pad146when the box is closed as shown inFIG. 13. The bores134and the rods136are aligned such that when an object holder27is placed in any bore134, the stem26contacts a rod136. When the moistening tray106is in an open position as shown inFIG. 12, the rods136are pulled upwardly in a direction toward the moistening pad146by the springs138that are attached to an upper portion of the structure132. In such an open position, the rods136seat in an upper notch152of the sliding bars140. When the lid124is manually closed as shown inFIG. 13, the lid124makes contact with the levers144, pressing the levers144in a downward direction, causing the levers to pivot about a pivot pin153and thereby press against the sliding bars140, causing the bars140to slide in a horizontal direction toward the stop151which in turn causes the rods136to slide along the curved surfaces142, expanding the spring and moving the rods downward toward the base120to a closed position seated in a lower notch154of the sliding bars140. When the lid124is completely closed, the object holders27are fully seated in the platform structure132with the undersides108thereof contacting the moistening pad146. When the lid124is opened, the tension on the rods136pulled by the springs138lifts the rods136and engaged object holders27upwardly, lifting the plates110of the object holders27up and away from the moistening pad146for easy grasping by a user. Furthermore, the spring-loaded construction of the structure130is flexible to provide for manual pressing of an object holder plate110down onto the pad146when the tray is in the open position shown inFIG. 12, thereby lowering the rod136and providing for re-wetting of the object holder underside108prior to installation of the object holder27on the cryodisc22. As shown inFIG. 12, the bores134are formed such that when the tray106is in the open position ofFIG. 12, the object holders27are elevated with the plates110slightly tilted, making the object holders27easier to grasp.

Preferably, the moistening pad146is saturated with 70% isopropyl alcohol. Furthermore, a sodium chloride solution may be added to the isopropyl alcohol, and the resulting mixture utilized to saturate the moistening pad146. When an object holder27wetted with the isopropyl alcohol/sodium chloride mixture is placed onto a frosted linear cryodisc22, the linear motion platform12elevates and contact is made between the lower, wetted surface108of the object holder27and the upper surface of the frosted cryodisc60. Advantageously, consistent and extremely rapid heat transfer occurs between the tissue object holder27and the cryodisc22.

As previously discussed herein, tracking of tissue specimens17during processes according to the invention is aided by engraving or otherwise placing coordinating identifying numerals on and near the cryodiscs16and22. Further tracking may be accomplished by the use of a labeling system that includes small discs with micro numbers and/or letters and/or corresponding bar codes, shown as micro-labels160in FIGS.1and14-17. Each micro-label160is packaged with accompanying identically numbered or otherwise marked macro-labels or dots that may be secured by an operator to the name area of an examination slide as well as to report and log books. A bar code or a set of numbers and/or letters are embedded throughout a full thickness of the label160so that when the frozen specimens17are sliced, a slice of precise information also accompanies the tissue specimen onto a glass examination slide162as illustrated inFIG. 17. When the microscopist examines the tissue specimen17, the micro-label160with a number, letters or bar code is visualized and manually recorded or may be recognized by a scanner. For multiple specimens from one patient, discs may be available that represent different sub-units. Such can be detected, for example, by using lower case letters or numbers with different fonts. It is foreseen that such micro-labels160may be utilized with permanent tissue specimens as well as with the frozen specimens17according to the present invention. The micro-labels or discs160are preferably made from a protein substance that adheres well to glass slides and more preferably of a stained protein using silver, using known micro-printing technology with ink preferably reinforced with silk or other protein, such as that found in spider webs, to provide stability during slicing and viewing. Moisture barrier packaging for the micro-labels160is preferred that maintains the labels160in a desired moisture range for ease in slicing. Macro-labels may be packaged adjacent the protein disc160and be of sufficient quantity to label glass slides, reports, and/or log books.

During a process according to the invention, which will be described in greater detail below, tissue specimens17are initially frozen on the rotary platform cryodiscs16and then preferably covered with a viscous, fiber-reinforced embedding medium170, followed by transfer the to object holders27disposed on the linear platform cryodiscs22. Furthermore, an amount of a standard embedding medium, such as O.C.T. compound (not shown), is preferably placed on the object holders27, filling in the grooves114thereon prior to transfer of the frozen tissue specimens17onto the object holder27. O.C.T. is an abbreviation for “Optimal Cutting Temperature.” O.C.T. is a well-known water soluble embedding medium for frozen tissue specimens, and for example, is sold under the mark TISSUE-TEK® by Sakura Finetechnical Co., Ltd., Tokyo,103, Japan. The TISSUE-TEK® O.C.T. compound includes 10.24 weight percent polyvinyl alcohol; 4.26 weight percent polyethylene glycol; and 85.50 weight percent non-reactive ingredients (water).

As shown inFIG. 15, an applicator tube172is used to manually apply the fiber-reinforced embedding medium170onto the specimens17. The embedding medium170is useful for holding the specimen17in place during slicing and transfer of a sliced specimen173to a glass slide162for review. A feature of the fiber-reinforced embedding medium170is that it adheres well to tissue specimens17and does not chip or break into chunks during the specimen slicing process.

A preferred fiber reinforced embedding medium for use according to the invention includes a mixture of (1) a known polyvinyl alcohol/polyethylene glycol embedding medium, such as the TISSUE-TEK® O.C.T. compound previously described herein; (2) fiber; and (3) an electrically conductive polymer. A particularly preferred embedding medium includes mixing the TISSUE-TEK® O.C.T. compound and the following: protein, preferably in the form of a silk fibers; cellulose, preferably bamboo cellulose; and polyaniline, preferably charged polyaniline. Another polyaniline for use according to the invention is a long chain polyaniline emeraldine salt grafted to lignin in a 20 wt. % dispersion in water (available from Sigma-Aldrich Corp., St. Louis, Mo.). It is believed that electrically conductive polymers such as polyaniline cross link with the cellulose fibers, forming a matrix or lattice therewith, providing control, hold and strength to the embedding medium. The following Example discloses a particularly preferred fiber-reinforced embedding medium170.

EXAMPLE

DensityApproximateComponent(g/l)Amounts (g)Silk Fibers0.1 to 5QC 200 Fiber1>554QC 150 Fiber1>1601QC 90 Fiber1>1701Ball ® Fruit Jell ® Pectin221QC Fiber is a product designation for bamboo-fiber available from CreaFill Fibers Corp., 10200 Worton Road, Chesterown, MD. The numerals 200, 150, and 90 designate grades, primarily indicating density and particle size ranges.2Registered trademarks owned by Jarden Corp. for powdered mixture of sucrose, dextrose and citric acid.
The above ingredients were mixed and folded into the following mixture at 60° C.:

To enhance adhesion between the tissue specimens17and the fiber-containing embedding medium170, it is helpful to warm the applicator tube172filled with the medium170on a heating device, such as the heated roller mixer174shown inFIG. 18. The device174is generally of known construction in the cooking arts and includes a heating element176disposed beneath rotating rollers178. Preferably, the roller mixer174mixes the medium170and heats the medium170to a temperature range of 25° C. to 65° C.

A process for the surgical removal of tissue specimens and preparation for histologic examination according to the invention is set forth in the following steps. Although the process is described with respect to a single tissue specimen17, it is noted that for the illustrated embodiment of the apparatus1, up to twelve specimens17may be processed during a single cycle of the apparatus1. Initially, a tissue specimen17is excised as a truncated sphere; desirably including an entire tumor to be removed. The specimen17is then relaxed by cutting on the skin side, which is a partial sphere, to relax and flatten the specimen. Next, four corners of the specimen17are marked with blue, green, red and orange dye to make it possible to identify the orientation of the specimen17relative to the patient's wound. A description or other record is kept of the orientation of the wound with respect to the patient and the specimen17and where the dye is placed.

The relaxed and dyed specimen17is then placed on the surface21of a rotary platform cryodisc16with the skin side facing up and away from the surface21. A micro label160is placed next to the specimen17. Plastic film20is then placed over the specimen17and the micro label160is moved and pressed against the specimen17, with the specimen17being manually urged to a substantially flat orientation on the surface21. The film20is also manually pressed against the adhesive filled groove19. A vacuum is drawn through the groove18to about 27 mm Hg under the film20to snug the specimen17and accompanying micro label160against the surface21of the cryodisc16. With reference toFIG. 14, such a vacuum process is shown being applied to two specimens17on each cryodisc16, each with an adjacent micro label160and covered with a plastic film20.

A quantity of cryogenic fluid is then circulated throughout the fluid transfer system3in the rotary and linear cryodiscs16and22, as previously described. Circulation of the liquid chills the rotary discs16to a preferred temperature, that varies depending upon the preference of the technician, but is typically between about −30° C. and about −50° C., and more preferably between about −40° C. and about −50° C. It takes between about thirty to about forty-five seconds for the discs16to cool to the desired temperature.

When the specimen17starts to freeze, which is determined visually by change in appearance, the film20is carefully peeled away. Then, fiber reinforced embedding medium170is squeezed from a tube172directly and firmly against and around the frozen specimen17and the micro label160.

The object holder moistening tray106is then opened and an object holder27having an underside108wetted with isopropyl alcohol or an isopropyl alcohol sodium chloride mixture, is placed on the cryodisc22. A light or very thin coat of less viscus embedding media, such as the standard embedding medium (O.C.T. Compound) previously described herein, is placed on the object holder plate110to fill grooves114therein. Alternatively, the standard embedding medium is applied on top of the fiber reinforced embedding medium170that covers the specimen17.

The moistened object holders27are placed in respective bores24with the object holders27somewhat spaced from the surface of respective cryodiscs22until the platform10or11is rotated from a position lateral to the platform9to a covering position and the respective cryodisc or cryodiscs16with the frozen specimen or specimens17thereon each engage a respective object holder27at which time the engaged object holder27drops and comes in touching contact with the top surface60of the cryodisc22. In this manner, the object holder27becomes a near room temperature object holder engaging the cold specimen17which warms near the engagement and then is again quickly recooled when the object holder27engages the cryodisc22. This aids in adhesion between the specimen17and the object holder27, while the isopropyl alcohol cooperates with frost on the coating of the surface of the cryodisc22to enhance heat transfer and provide rapid and consistent cooling to the object holder27.

The temperature of the combined object holder27and specimen17is adjusted according to the preference of the technician. The object holder27and specimen17are then placed in a slicing apparatus and sliced. Often ten to fifteen slices at about four microns each are taken until the technician is satisfied that the entire surface to be studied is represented.

Some technicians remove the embedding media170from the specimen17during slicing and then transfer the slice173to the slide162by means of an anti roll plate179. The anti roll plate179helps prevent curling or rolling up of the specimen. Some technicians do not use the anti roll plate179and prefer to use the embedding media to drag the specimen slice173to the slide162. In order to do this, the media must adhere to the specimen. This is accomplished by heating the fibrous embedding medium170first to between about 35° C. to about 65° C. on the heater174. When the medium170touches the specimen17there is a quick thaw and then re-freezing that adheres the medium170to the specimen17. In such case, the embedding media170serves a second transfer function in addition to the function of stabilizing the specimen17during slicing.

The specimen slice173is then examined by viewing the tumor margin surface area under a microscope. If tumor tissue is found in the studied slice173, another tissue specimen is harvested and the process is repeated until the margins of the specimen17are completely free of tumor.

With particular reference toFIG. 15, a technician may prefer to place a relaxed and inked tissue specimen17on a small sheet of a polyester film, for example, a polyethylene terepthalate film sheet182, such as provided under the trademark MYLAR® by Dupont Tejjin Films, to initially hold the specimen17on the rotary cryodisc16, rather than placing the specimen17directly on the cryodisc16. It is foreseen that an electrostatic (+) charged film, such as plasticized vinyl film, may also be used. The polyester film sheet182preferably has a thickness of 3 mils (0.003 inches equivalent to 0.0765 millimeters) and includes a slightly roughened surface finish on one side184thereof, suited for printing a grid or map thereon, and an opposite smooth side. The sheet182may include a pre-printed grid pattern or tissue orienting pattern as described more fully below with respect to a temporary tattoo according to the invention, including horizontal, vertical and radial lines with numerals identifying horizontal, vertical and radial locations and certain grid portions and numerals being printed in color, also as described with respect to the temporary tattoo. Additionally, the sheet182may include numerals to aid in the identification of the samples that coordinate with the numerals engraved or otherwise printed on the platforms10and11and the cryodiscs16and22. The grid may be printed on the polyester sheet182with an ink or other substance similar to what is used for temporary tattoos, so that the grid pattern is transferable to the moist specimen surface, as described more fully below. Because the grid pattern and reference numeral printable substance does not readily transfer onto fat, preferably the reference numerals are repeated across a length of the grid design on the polyester sheet to ensure identification of a top surface of the frozen specimen with a reference numeral coordinating with the numerals engraved or otherwise printed on the platforms10and11and the cryodiscs16and22.

The sheet182may be sized to fit on a cryodisc16such that all three specimens fit on a single sheet182. In such a situation, three grid patterns may be printed on a single sheet182. Alternatively, smaller sheets182may be used, one for each specimen17, as illustrated onFIG. 15. In such a situation, a single grid pattern, such as that shown inFIG. 19, may be printed on each sheet182. Furthermore, for certain procedures requiring large specimens, a larger sheet182may be used that is printed with a single grid pattern, similar to the pattern shown inFIG. 19. In such a usage, because the sheet182is larger, more detailed color coding and additional identifying numerals may be printed with the horizontal, vertical and particularly with respect to the radial grid lines, further aiding the process of locating and identifying tumor margin surfaces of interest. In particular, numerals may be printed about a circumference of a circular design having a plurality of horizontal and vertical lines and identifying radial lines with respect to a color coded X and Y axis (as described herein with respect to the pattern190shown inFIG. 19). The segments formed by the radial lines are identified by numerals printed about the circumference of the design in both degrees and in the clock-wise fashion described herein with respect to the pattern190shown inFIG. 19. Depending on the size of the sheet182, the radial segments may be identified along the design circumference in segments of fifteen degrees each, corresponding to thirty minute increments. The numerals located about the design circumference may also be printed or shaded with the same color coding as described herein with respect to the X and Y axis ofFIG. 19.

Preferably, the sheet182is prepared for use according to the invention by brush coating with a natural protein, such as egg albumin on the slightly roughened and possibly printed surface184. It is foreseen that other types of materials that have tissue adhesive or affinity properties may be used as coating material, including poly-L-lysine, aminoalkysilane, glycoproteins and gelatin. Although not required, a technician may also desire to score the opposite, smooth side of the sheet182, most preferably with some of the scores extending completely through the sheet182. A drop or other small amount of the standard embedding medium previously described herein is first placed centrally on a section of the surface21of the cryodisc16where the specimen17will subsequently be placed. The sheet182is then placed on top of the standard embedding medium, with the smooth or scored side down and the tissue specimen17is placed on the side184of the sheet182coated with the albumin. The specimen17is then manually pressed against the sheet182, the specimen17advantageously adhering to the surface184. Alternatively, the specimen17is placed upon and pressed against the sheet182prior to placing the sheet182on top of the standard embedding medium on the cryodisc16. Then, if desired, the operator may lift up the sheet182and view the specimen17underside through the substantially clear sheet182, to ensure that edges of the specimen17are pressed against the surface184as desired by the operator. Photographs may also be taken of the specimen17through the film sheet182to further document and track the particular specimen. Thereafter, the sheet182with the specimen17thereon, are placed on the cryodisc16, with the standard embedding medium disposed between the sheet182and the cryodisc16to temporarily secure the sheet182to the cryodisc16during subsequent rotation of the cryodisc16over to an object holder27. Similar to the process steps previously described herein, a micro label160may be placed adjacent the specimen17and the specimen17, label160and sheet182are then covered with a plastic film20and vacuum is pulled, followed by freezing of the specimen17. The plastic film20is then removed and the fiber reinforced embedding medium170is applied on top of the frozen specimen17and accompanying label160as previously described herein. A standard embedding medium is then applied on top of the fiber-containing embedding medium170or, alternatively onto a selected, wetted object holder27, removed from the moistening tray106and placed on a cooperating cryodisc22. The embedded specimen17is then rotated and transferred to the object holder27on the cryodisc22as previously described herein. After transfer, a desired specimen temperature is reached and the polyester film sheet182is removed from the specimen17. When the film sheet182has a grid pattern printed on it, any pattern or identification numeral printed on the film sheet182advantageously transfers to a top surface of the specimen17, providing for additional identification and tracking as the specimen17is transferred from the cryodisc22to a slicing machine. The specimen17is then sliced and the sliced specimen173is reviewed.

In a process of the invention previously described herein, one of the method steps includes the marking of the specimen17with blue, green, red and orange dye to help identify the orientation of the specimen17relative to the patient's wound. With reference toFIGS. 19-21, such an orientation step is aided by the use of a tissue orienting pattern or grid illustrated as a temporary tattoo190inFIG. 19. The patterned temporary tattoo190is placed on the tumor-containing area of skin192of the patient194, prior to harvesting the tissue specimen17. The pattern190according to the invention shown inFIG. 19includes an X axis196, a Y axis198and a plurality of concentric circles200. The circles include radial marks or ticks202. The tissue orienting pattern190is further marked in a clock-wise fashion with a numeral “12” at a top204of the Y axis; a numeral “3” at an end location206on the X axis ninety degrees from the numeral “12”; a numeral “6” at a bottom208of the Y axis; and a numeral “9” at an end210of the X axis ninety degrees from the numerals “6” and “12” The X and Y axes of the pattern190are in color, with the Y axis portion extending from the center212to the numeral “12” being blue; the Y axis portion extending from the center212to the numeral “6” being in green; the X axis portion extending from the center212to the numeral “3” being in red; and the X axis portion extending from the center212to the numeral “9” being in orange. This color combination corresponds to the following standard practice of clock-wise marking of a specimen: blue at twelve o'clock; red at three o'clock; green at six o'clock; and orange at nine o'clock.

As illustrated inFIG. 21, after a tissue specimen17is harvested, a portion of the pattern190remains on the patient194, clearly showing the X and Y axes,196and198, respectively, and remaining concentric circles200. The harvested specimen17is then inked at a top, bottom and either side thereof consistent with the tattoo190and is then frozen, sliced and examined as previously described herein. If further tissue removal is necessary, the tattoo pattern190remaining on the patient194then provides an accurate map for reorienting the specimen17with the patient and removing tissue from a desired area.

The pattern190may also be imprinted on the printable side184of the polyester sheet182. The pattern or grid190allows a technician to precisely locate tumor roots on the tissue specimen17as the specimen17is pressed onto the sheet182. Notations may then be made to allow for greater accuracy during reorientation after a respective specimen slice173is microscopically reviewed.

In a further aspect of the invention, known software may be utilized to create a digital contoured map of a floor and walls of a specimen17in order to improve accuracy during reorientation. For example, after a temporary tattoo190is applied to a patient194and a tissue specimen17is harvested, a first digital picture may be taken of the specimen17above the tissue and a second picture taken below the tissue, both pictures oriented with respect to the tattoo190. Then, a digital contour map is created for a periphery of the specimen17, digitally compensating for any relaxing and flattening of the specimen17. Pathologic notations made by the operator viewing the specimen slice173may be loaded into the compensated digital contour map. The compensated contour map is then digitally returned to the original specimen contour and then clinically reoriented to the tattoo190on the patient's wound area192, indicating with greater accuracy the pathologic notation taken from the sliced specimen173.

Temporary tattoos for use according to the invention are preferably polymer based. A preferred temporary tattoo190includes a polyvinyl alcohol resin mixed with linseed and mineral oils, obtainable from, for example, Johnson & Mayer, Inc., Hackensack, N.J.