Patent Description:
Tissue that is surgically removed from patients or subjects is routinely frozen for both diagnostic and research needs. There is a lack of standardization in freezing protocols, partially driven by utility. Tissue obtained from frozen sections for histopathologic evaluation under the microscope is routinely frozen in a freezing compound, the most commonly used is OCT (Optimal Cutting Temperature - a mixture of polyproplyglycols, sucrose and water). Tissue obtained for research, or destined for a biobank is routinely frozen in the same matrix. This matrix has the benefit of protecting the tissue, as well as functioning as an embedding matrix for the preparation of frozen sections on a cryotome.

Currently, there is no routine method of storage of OCT embedded tissue after freezing. Small molds may help position and orient the tissue, but a protective enclosure that can be labeled and does not require the tissue to be partially thawed for use (cryosection, TMA production, other sampling) does not exist.

<CIT> discloses a tissue cassette, comprising a base including side walls and a bottom wall that define a chamber adapted to receive a tissue sample, and a lid provided to the base and adapted to cover the chamber to retain the tissue sample within the chamber.

According to the present invention, there is provided a frozen tissue specimen storage kit comprising:.

Also disclosed herein, but not claimed, is an article comprising:.

Further disclosed herein, but not claimed, is a method for preparing a frozen tissue specimen block comprising:.

Additionally disclosed, but not claimed, is a method for microtome sectioning a frozen tissue specimen block comprising:.

The foregoing will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.

Disclosed herein are devices and methods for freezing and storing tissue samples, particularly biological tissue samples that are prepared for cryosectioning, archiving as frozen tissue, or subsequently fixed and processed as surgical specimens. Currently, the handling, labeling and storage of OCT-embedded frozen tissue is complicated, with ad hoc solutions applied, and no standardization. The vexing issue, well documented in the literature, is the temperature fluctuation that occurs in frozen tissue handling, resulting in specimen degradation. A second, but equally problematic issue is specimen labeling. No current solution allows for a durable label to be affixed to the OCT embedded specimen. Disclosed herein is an insulated storage unit, with integrated labeling, that addresses these problems.

In general, a tissue specimen is placed in a mold and a freezing matrix material (e.g., OCT) is applied to the tissue sample via introduction of the freezing matrix material into the mold. The freezing matrix material may fully or partially encompass the tissue sample. The resulting matrix material-embedded tissue is secured or placed onto a base (via a Slotted U-shaped clamp). The mold/base/tissue sample is then frozen (along with Slotted U-shaped clamp). In certain embodiments, the base and a cooperating lid are snapped shut, and the entirety of the base/tissue sample/lid unit is then frozen. Freezing may be accomplished by a variety of methods including placing the unit in a freezer, placing the unit in freezing bath (e.g., isopentane, liquid nitrogen, dry ice (with or without an ethanol slurry), or placing the unit on a frozen surface. The freezing process secures the base to the frozen block of freezing matrix material and tissue. After freezing, the mold can be removed (the lid can also be removed). The base is coupled to a chuck, meaning that the chuck is mounted with the frozen tissue available for immediate cryosectioning. After cryosectioning, the lid is replaced and the specimen is placed in an appropriate freezer. Alternatively the unit can be immediately archived for future use.

In a further implementation, after cryosectioning the lid is placed onto the base, and the resulting unit is immersed in a fixative, such as neutral buffered formalin or other solutions at room temperature. Although the freezing matrix material will dissolve, the tissue specimen is retained in the orientation that it was cryosectioned. This alignment facilitates the production of well-aligned permanent section for evaluation by a pathologist for diagnosis confirmation after rendering a preliminary frozen section diagnosis.

A mold is provided for embedding a tissue specimen in a matrix material. The matrix material may be any material suitable for freezing while maintaining the integrity of the tissue specimen. Illustrative matrix materials include water, saline, honey, sucrose and other sugar solutions, including those that contain formaldehyde, polyethylene glycol solutions. Freezing media include compounds of: saline solutions; sugar solution, most commonly sucrose and trehalose; polyethylene glycols; cellulose agarose; and latex. The matrix material may contain other chemicals, including fixatives, such as ethanol, formaldehyde, acids (acetic and picric for examples) and other chemical solutions that undergo a phase transition from gel(viscous liquid) to a solid under change of temperature (typically cooling, however heating (hydrogel) Particular matrix materials are optimal cutting temperature compound (OCT, e.g., Tissue Tek™ available from Sakura Finetek, Jung Tissue Freezing Medium, or Leica Microsystems Surgipath FSC <NUM> Frozen Section Compound). The mold may be any mold suitable for holding a tissue specimen and the matrix material. An example of a mold <NUM> is shown in <FIG>. The mold <NUM> includes a cavity <NUM> into which a tissue specimen <NUM> is placed. The mold can have differing depths and shapes. For example, some molds are round. Typical round molds have a <NUM>-<NUM> diameter, and a <NUM>-<NUM> depth. Typical rectilinear molds are <NUM>-<NUM> on sides, and <NUM>-<NUM> depth. A liquid matrix material <NUM> is introduced into the mold cavity <NUM> to partially or fully encompass the tissue specimen <NUM>. The mold may include a flange <NUM>, or a similar element, for handling and positioning the mold as required. In certain embodiments, the mold is made from a flexible material such as, for example, clear plastic.

A base then is aligned and placed on the mold that contains the tissue specimen and the matrix material to form a mold/base construct. An example of a planar base <NUM> is shown in <FIG>. The base <NUM> includes a first surface <NUM> and an opposing second surface <NUM>. The base <NUM> also defines an outer peripheral edge <NUM>. The base <NUM> further includes protuberances <NUM> extending from the second surface <NUM>. The protuberances <NUM> may be in the form of an array of individual small bumps or small projections. The protuberances array may be centered on the second surface <NUM>. The protuberances assist in holding the tissue specimen in place.

The base <NUM> further includes a ridge <NUM> at the peripheral edge <NUM> on three sides of the base. In certain embodiments, the ridge <NUM> could be provided on two sides or four sides of the base. In certain embodiments, if a circular mold is used the ridge <NUM> could be a matching circular shape. The ridge <NUM> is low in height (e.g., <NUM> to <NUM>) and can be <NUM>-<NUM> in width and does not constitute laterally extending walls. The mold <NUM> fits into the base <NUM> as shown in <FIG> such that the peripheral edges of the mold flange <NUM> contact the ridge <NUM> on multiple sides (e.g., three sides) of the base <NUM>. Thus, the ridge <NUM> retains the mold <NUM> forming a mold/base construct <NUM> as demonstrated in <FIG>. The second surface <NUM> of the base <NUM> contacts the mold <NUM> so that the second surface <NUM> covers the mold cavity <NUM>. In particular, the base protuberances <NUM> are aligned with and contact a layer of matrix material disposed on top of the tissue specimen <NUM>. The edge <NUM> of the base <NUM> that does not include a ridge receives an optional extension <NUM> of the mold flange <NUM>. An opening <NUM> is present in the ridge <NUM> opposite the edge <NUM> of the base <NUM>. The opening <NUM> provides a means for un-snapping the base from other components as described in more detail below. The base <NUM> also includes a plurality of holes <NUM> extending from the first surface <NUM> to the second surface <NUM>. The holes <NUM> permit discharge of excess freezing matrix material when the assembly is placed in a slotted U-shaped clamp as described below. In certain embodiments, the base does not include any holes.

In another embodiment of the base <NUM>, the ridge <NUM> is present on all four sides of the base. In this embodiment, the mold fits inside the ridges <NUM>.

The base also include an area <NUM> for labeling (shown in <FIG> and <FIG>). For example, a label identifying the specific specimen may be affixed to the facing-out first surface <NUM>. Illustrative labeling includes mechanical engraving or inscribing, writing in pencil or solvent resistant pen or printer, affixing an adhesive label (including a barcode), or affixing or embedding an RFID tag. Since the base travels with the specimen during processing, the label ensures that chain-of-custody for the specimen is maintained.

As shown in <FIG> a slotted U-shaped clamp <NUM> is used for securing the mold to the base during freezing. The slotted U-shaped clamp <NUM> includes a slot <NUM> and an open end <NUM>. The base <NUM> and mold <NUM> (i.e., the mold/base construct <NUM>) are together slid into the open end <NUM> of the slotted U-shaped clamp <NUM> via inserting the base <NUM> and mold <NUM> together into the slot <NUM> provided in the slotted U-shaped clamp <NUM> and the base <NUM> and mold <NUM>. In certain embodiments, the slot <NUM> may be offset from the vertical center of the U-shaped clamp <NUM> resulting in relatively flat mold/base construct <NUM>. For example, the slot <NUM> may be <NUM> from one surface of the U-shaped clamp <NUM> and <NUM> from the opposing surface of the U-shaped clamp <NUM>. In certain embodiments, the mold extension <NUM> extends beyond the open end <NUM> of the slotted U-shaped clamp <NUM> to provide a means for holding the mold and base while inserting and removing the mold and base. In other embodiments, the base has a sufficient length to enclose mold extension <NUM>. In certain embodiments, the slotted U-shaped clamp can accept the mold extension into the clamp to provide more surface area for clamping. The slotted U-shaped clamp <NUM> holding the mold/base construct <NUM> (see <FIG>) then is subjected to a temperature sufficient for freezing the matrix material and tissue specimen resulting in converting the tissue specimen block into a frozen tissue specimen block <NUM> (block <NUM> is shown in <FIG>, <FIG>). The temperature depends upon the specific matrix material. The freezing temperature may range, for example, from <NUM> to -<NUM>, more particularly <NUM> to -<NUM>. The freezing, base protuberances <NUM> and/or base holes <NUM> are sufficient for securing the matrix material and tissue specimen onto the second surface <NUM> of the base. After formation of the frozen tissue specimen block <NUM>, the mold/base construct <NUM> is removed from the slotted U-shaped clamp <NUM> as shown in <FIG>.

After formation of the frozen tissue specimen block, the mold is removed from the base. The mold is a smooth material that is sufficiently flexible, even at low temperatures, and it is snapped, peeled, or pulled off. An example is shown in <FIG>. The mold <NUM> can be removed by grasping the mold flange extension <NUM> and separating the mold <NUM> from the base <NUM>. After removal of the mold, the tissue specimen block <NUM> extends from the second surface <NUM> of the base <NUM>. In certain embodiments, the tissue specimen block <NUM> is centered on the second surface <NUM> of the base <NUM>. The tissue specimen block <NUM> also is supported in a standalone position on the second surface <NUM>. In other words, the tissue block <NUM> is not surrounded by, or retained by, any external walls.

The base holding the tissue specimen block may be coupled with a lid to form a base/lid construct <NUM> (see <FIG>, <FIG>). In certain embodiments, the lid and base are snapped together. Base/lid construct <NUM> is for storage of the frozen tissue block in a controlled environment.

In certain embodiments, the base holding the tissue specimen block may be attached to a chuck that is, in turn, attached to a cryostat as described in more detail below. The tissue specimen may then be cryosectioned prior to coupling of the lid.

An example of a lid <NUM> is shown in <FIG>. The lid <NUM> has a first side <NUM> and an opposing second side <NUM>. The first side <NUM> defines a continuous planar surface. The second side <NUM> defines a recessed portion <NUM> configured to cover the tissue specimen block. The recessed portion <NUM> includes a bottom <NUM> and laterally extending walls <NUM>. In <FIG> the frozen tissue specimen block <NUM> does not contact the bottom <NUM> or walls <NUM> of the recessed portion <NUM>.

The lid <NUM> also includes an outer rim <NUM> configured to engage with the outer peripheral edge <NUM> of the base <NUM>. In certain embodiments, the outer rim <NUM> and the peripheral edge are snap-fit together such that the lid <NUM> and the base <NUM> are can be coupled and de-coupled from each other. The outer rim <NUM> does not form part of the recessed portion <NUM> due to presence of a shelf <NUM> disposed between the outer rim <NUM> and the walls <NUM> of the recessed portion <NUM>. In certain embodiments, the outer rim <NUM> extends upwards from the shelf <NUM>. The peripheral dimension of the outer rim <NUM> is such that the peripheral edges <NUM> of the base <NUM> fit within the outer rim <NUM>. In certain embodiments, the shelf <NUM> defines a slot <NUM> located contiguous to the outer rim <NUM> and that engages with the ridge <NUM> of the base <NUM>. In certain embodiments the outer rim <NUM> is present on only three peripheral sides of the lid <NUM> that coincide with the three peripheral sides of the base <NUM> at which the ridge <NUM> is also present. Thus, the lid <NUM> is removably coupled to the base <NUM> via a snap-fit constructed between (a) the outer rim <NUM> of the lid <NUM> and the peripheral edge <NUM> of the base <NUM> and (b) slot <NUM> of the lid <NUM> and the ridge <NUM> of the base <NUM>.

One side <NUM> of the lid <NUM> does not include the outer rim <NUM>. An opening <NUM> is present in the outer rim <NUM> opposite the side <NUM> of the lid <NUM>. The location of lid outer rim opening <NUM> coincides with the location of the base ridge opening <NUM>. Openings <NUM> and <NUM> cooperate to provide an opening for insertion of a fingernail or a mechanical lever for un-snapping the base <NUM> from the lid <NUM>.

Another embodiment of a lid and a base is shown in <FIG>. In this embodiment, the base includes a single, centered hole <NUM> that permits exiting of excess freezing matrix material. The lid includes a plurality of holes <NUM> extending through the recessed portion bottom <NUM>. The holes <NUM> allow access for a liquid fixative into the base/lid construct holding a tissue specimen, for example, when the construct is immersed in the liquid fixative.

The final assembly shown in <FIG>, <FIG> stores the frozen tissue specimen block <NUM> between the base <NUM> and the lid <NUM>. The final assembly also may have an outside surface area for labeling, for example, written, barcode (printed), adhesive label, or an integrated RFID tag. The closed assembly protects the tissue specimen from deformation and thermal isolations and shock, within the freezer (small changes in temp), during handling, inventory and management (moving from freezers), as well as provides insulation when in transfer. The fact that the lid can be removed while the base remains in a freezing compartment or on a frozen plate/surface, and the base affixed and cut, without melting/and embedding means the tissue does not have to have a freeze-thaw cycle to cut new sections after storage. The assembly is modular so that it will fit in existing storage units commonly used in research and clinical settings, with the label readily presentable. In certain embodiments, the dimensions of the final assembly are sufficiently large to hold a whole body organ (e.g., primate brain, kidney, etc.) cross-section. In certain embodiments, the dimensions of the final assembly may be <NUM> to <NUM> in length, <NUM> to <NUM> in width, and <NUM> to <NUM> in thickness. In certain embodiments, the dimensions may be <NUM> x <NUM> x <NUM>. In certain embodiments, the dimensions may be up to <NUM> x <NUM> x <NUM>. In certain embodiments, the assembly is affordable so that it can be a single use object. The modular device disclosed herein is designed to work with any tissue holders on cryostats or freezing mold models.

The lid and base may be made from any suitable material. The material should be temperature-stable (i.e., insulative), resist solvents such as xylene or alcohols, and resist fixatives such as neutral buffered formalin. Acrylonitrile butadiene styrene (ABS) and polyamide (e.g., nylon) are illustrative plastics for making the device.

In certain embodiments the base that holds the frozen tissue specimen may be attached to a chuck that is, in turn, attached to a cryostat. For example, as shown in <FIG>, a chuck <NUM> is provided with an arm <NUM> for mounting the chuck onto a cryostat. The arm <NUM> may be in the form of a cylinder or shaft that extends from a first surface <NUM> of the chuck <NUM>. The chuck <NUM> includes a second surface <NUM> opposing the first surface <NUM>. The base <NUM> may be attached to the second surface <NUM> of the chuck <NUM>. For instance, the second surface <NUM> may define a peripheral raised rim <NUM> that is dimensioned to receive the base <NUM>. The first surface <NUM> of the base contacts the second surface <NUM> of the chuck <NUM>. In certain embodiments, the raised rim may include at least one opening <NUM> into which a fingernail or mechanical lever can be inserted for removing the base from the chuck. The lid <NUM> is removed from the base <NUM> thus making the frozen tissue specimen available for cryosectioning.

An illustrative process is described below:
A thin layer of freezing medium is usually applied to the bottom of the well of the cryomold. The tissue is then placed in the mold. The freezing matrix material is then added around the tissue until the mold is full, being careful not to displace the tissue when filling the mold. The open side of the mold is up as to not spill the freezing matrix material. Place the base structured side down, the edges of the base align it to the freezing mold. At this point wipe off extraneous freezing media. Slide the mold and base into the slotted U-shaped clamp and freeze. After freezing is complete any excess freeing media should be scraped off. Once frozen the rough surface of the base will secure the frozen block of freezing medium and tissue to the base. The cryomold can now be removed and discarded.

The articles and methods disclosed herein will facilitate specimen handling where the frozen tissue goes from a cryostat to a tissue processing cassette without manipulation. There is an incentive for this, as it will preserve the "cut face" of the tissue to allow better matching of the frozen sections to the permanent sections, a critical and complicated aspect of quality assurance in surgical pathology.

Claim 1:
A frozen tissue specimen storage kit comprising:
a base (<NUM>) having a first surface (<NUM>) and an opposing second surface (<NUM>) and an outer peripheral edge (<NUM>), wherein the second surface includes an area comprising a plurality of protuberances (<NUM>) and configured to attach and maintain orientation of a frozen tissue specimen block;
a tissue specimen mold (<NUM>) aligned with the area of the second surface of the base;
a removable lid (<NUM>) having: (i) a first side (<NUM>) and a second side (<NUM>), wherein the first side defines a continuous planar surface and the second side defines a recessed portion (<NUM>) configured to cover a frozen tissue specimen block; and (ii) an outer rim (<NUM>) configured to engage with the outer peripheral edge of the base;
a slotted U-shaped clamp (<NUM>) configured to receive the base and the tissue specimen mold; and
a chuck (<NUM>) configured for mounting at least the base onto a cryostat.