WO2018195353A1

WO2018195353A1 - High-throughput histopathology tool and methods of use

Info

Publication number: WO2018195353A1
Application number: PCT/US2018/028433
Authority: WO
Inventors: Lan Wei; James H. LU
Original assignee: Biolyceum, Llc
Priority date: 2017-04-20
Filing date: 2018-04-19
Publication date: 2018-10-25

Abstract

A method of preparing a histopathology sample using a container with an internal volume. A divider is placed in the container so as to divide the internal volume into separate compartments. A tissue specimen is placed in at least one of the compartments. Paraffin or another embedding medium in a liquid state is added to the internal volume of the container such that the divider material and the specimen are at least partially immersed in the medium. The medium is then allowed to solidify, to create a unit. The unit can then be sliced along a plane that intersects the specimen, the divider material, and the medium to create a thin section of the unit that comprises the specimen, the divider material, and the medium, and that is adapted to be observed using a microscope or other observation equipment.

Description

High-Throughput Histopathology Tool and Methods of Use

BACKGROUND

[0001] This disclosure relates to a high-throughput embedding, sectioning, and data analysis system that is used in histopathology, which is a process of preserving the microstructure and genetic materials of biopsy/necropsy taken from patients or animals for analysis of changes in disease.

[0002] Histopathology is the current standard of diagnosis and prognosis for healthcare and pharmaceutical drug discovery. The conventional formalin fixing and paraffin embedding (FFPE) process is widely used because of its superb ability to preserve native fine structure of tissues for extended periods at room temperature. However, it is labor intensive and time consuming, involving tedious steps including tissue processing, paraffin embedding, microtome sectioning, histo-/immuno-staining, and data analysis. Typically, tissues from patients or animals are fixed in formalin and dehydrated by gradually replacing water in the tissues with ethanol, then clearing with xylene. Next, tissues are embedded in paraffin or other specimen-embedding media like plastic. The paraffin block containing the tissue is sectioned by microtome into thin slices, and each slice is placed on a glass slide. The paraffin blocks and slides can be stored at room temperature for years before further examination. The tissues on glass slides can be stained using a variety of staining methods to reveal a general structure (hematoxylin and eosin [H&E], sirius red, trichrome, etc.), or a particular protein (immunohistochemistry [IHC], immunofluorescence [IF]), or RNA/DNA (in situ hybridization [ISH], fluorescent in situ hybridization [FISH], tunnel staining, etc.). Stained slides, or their digitalized images, are examined by pathologists to determine the degree of disease. Part of the tissue, or even a single cell, can be removed to examine at its molecular level, using methods such as microarraying, RNA sequencing, deep sequencing, exonal sequencing, proteomics, etc.

[0003] Many innovative methods have been attempted to increase the efficiency of FFPE process, focusing on staining and data acquisition/analysis. However, the efficiency of the entire process remains low with the treatment of each individual tissue sample. Several steps in the FFPE process have been improved and made more efficient, such as the automating of tissue dehydration or rehydration, slide staining, and differential labeling of multiple antibodies for simultaneous detection of multiple proteins. In addition, quenching/stripping methods have been utilized to allow a slide to be used more than once. High-resolution, whole-slide scanning has provided a digitized image of stained tissue, allowing the digital image to be analyzed and quantified by pathologists locally or around the world. This has allowed multiple reviews in determining the degree of disease within the tissue being evaluated. Thus, most of the steps within the FFPE process have been improved except the embedding and sectioning steps.

Presently, these are heavily labor intensive and inefficient with single tissue sample within a single paraffin block. Increasing the samples per block would greatly improve efficiency, however keeping tissues separated and easily identifiable are of the utmost importance. An efficient method of separating and tracking multiple tissues in a single block is needed. This disclosure is in response to this need.

[0004] Figure 1 illustrates steps in a common process 10 of a prior art of formalin fixing and paraffin embedding (FFPE) histology operation. This process includes: tissue

processing/dehydration 12; paraffin embedding 14; paraffin unit sectioning and placement 16, 18; tissue rehydration 20; staining 22; data acquisition and analysis 24; and molecular and genomic analysis 26, if needed. The embedding and sectioning steps 14, 16, 18 are time and labor intensive, while the efficiency of many other steps have been optimized through some degree of automation. If multiple tissue samples could be arranged in the embedding step 14, all them could be processed at the same time and oriented appropriately so the following steps 16- 26, may be as efficient as possible.

SUMMARY

[0005] The traditional FFPE histopathological process is labor-intensive and time-consuming albeit its pivotal role in disease diagnosis. It is heavily used to process patient biopsies in hospitals and to process animal samples from preclinical studies in pharmaceutical companies. The present high-throughput systems and methods greatly increase the cost-effectiveness of this process by utilizing a divider to separate multiple tissue samples in well-defined and trackable compartments. This divider can be sectioned and remain together with their tissues in all the downstream steps. The divider allows the separate tissues to be tracked and can also serve as a frame for automatic analysis of these tissues in digital images.

[0006] The system and method utilize the current histological equipment and reagents, but it revolutionizes the whole industry. Currently, the most limiting step in FFPE is sectioning, which heavily relies on the skill/labor of a technician. By using the present divider, the same time spent to process a paraffin block that normally contains a single tissue sample can be used to process multiple tissue samples. In addition, it reduces storage, material, reagents, and the time a pathologist switching between slides. Scientifically, it eliminates the technical variables during the FFPE process thus making results more comparable between samples. The divider sustains all the process of FFPE histology.

[0007] The system and method also contemplate a marker that can be inserted into tissues such as organs like mouse heart and lung. The marker stays in the tissue during processing and analysis and can help the pathologist to easily determine the orientation of the organ under the microscope. Another tool made of the same material can be used in MRI ex-vivo imaging of whole organ or tissue slices before histological analysis.

[0008] A series of dividers may be used to separate multiple tissues that are embedded within specimen-embedding media for the diagnosis and prognosis steps that follow. These dividers may be comprised of a variety of materials, such as silk, cotton, paper, or any other material with inert properties and purity and also are able to be sectioned by microtome. The material may be a natural material that comprises fibers or filaments. Silk, for example, has filaments composed of highly homogenous protein that allow the dividers to behave similarly to many specimen tissues. Paper on the other hand, has filaments composed of cellulose that does not stain the same as human/animal tissues. Like tissues, the dividers should be able to sustain heat and chemical solvents, should be able to be sectioned without damage to the microtome, should be able to remain on a glass slide, and should be able to be processed along with the tissues in further steps. The dividers may be used in diverse histology applications due to their nuclear-acid-free property and their extremely low reactivity to most antibodies used in immuno-staining. [0009] To keep track of tissues in a specimen-embedding medium block, the dividers can be arranged in an asymmetric fashion. In addition, pins or markers comprised of the divider material can also be used as reference points when inserted into tissue or alongside where orientation of tissue is important. All these reference points can be sectioned and maintain their relative position to tissues on slides. Thus, after sectioning, tissues may be easily identified by their positions relative to the divider or the inserted reference points. Furthermore, imaging software may utilize the divider as a frame for identification of tissue samples in the block.

[0010] The dividers reduce time, labor, material, and storage space. They also provide opportunities to directly compare tissues from control and treatment groups (or a single patient before and after treatment). Thus, the capacity of conventional histopathology can be greatly expanded. Furthermore, these dividers are preferably not magnetic, and therefore are useful for ex-vivo MRI imaging of multiple tissues before further histology analysis and for other non- histopathology applications.

[0011] In order for the tissue dividers to function properly, they should meet significant physical challenges beyond their ability to be immersed within paraffin. They should have the mechanical strength to stand on their own. They should be able to withstand near-boiling temperatures and they should be water-insoluble. The dividers should be able to be attached to a glass slide along with the tissues being analyzed and should be capable of being exposed to the chemicals used in FFPE histology, such as solvents like ethanol and xylene; fixative reagents like formalin; and other possible pH-changing reagents used in staining. In addition, the dividers should have a hardness that is similar to the tissues being embedded so that the microtome blade will not be damaged. The dividers should also contain no RNA or DNA to avoid interference with ISH, and they should have a low immune-reactivity to avoid interference with the IHC or IF. The subject tissue dividers possess all of these properties. Synthetic polymers or bio-gel extracted from animals may possess one or more of these properties, but not all of them and so may have less utility as divider material. For example, synthetic nylon and rayon cannot withstand some process temperatures and xylene. Bio-gel, mainly collagen extracted from pigskin, has properties of protein after fixation, but it may interfere with antibodies used in IHC. [0012] All examples and features mentioned below can be combined in any technically possible way.

[0013] In one aspect, this disclosure features a method of preparing a histopathology sample using a container with an internal volume. The method can includes the steps of placing a divider in the container so as to divide the internal volume into separate compartments, placing a tissue specimen in at least one of the compartments, adding to the internal volume of the container a medium that is in a liquid-state such that the divider material and the specimen are at least partially immersed in the medium, allowing the medium to solidify to create a unitary block, and slicing the block along a plane that intersects the specimen, the divider material, and the medium to create a thin section of the block that comprises the embedded specimen, the embedded divider material, and the medium. The section is adapted to be observed using a microscope or other observation equipment.

[00 4] The divider may adhere to glass, may be non-magnetic, may be resistant to ethanol and xylene, may be free of RNA and DNA, may be hyporeactive to antibodies, may be homogeneous, and may comprise silk, paper, spider silk, cotton, or a synthetic polymer, for example. The divider may be placed in an asymmetrical pattern so that the compartments are asymmetrical. The divider may comprise a plurality of separate cup-shaped internal volumes. The cup-shaped internal volumes may be formed. There may also be a marker in a compartment. The marker may be asymmetrical. The divider may comprise a porous bag. The porous bag may comprise a plurality of separate compartments.

[0015] In another aspect, this disclosure features a method of preparing a histopathology sample using a container with an internal volume, comprising the steps of placing a divider in the container so as to divide the internal volume into a plurality of separate compartments, wherein the divider comprises silk and adheres to glass, is non-magnetic, resistant to ethanol and xylene, free of RNA and DNA, hyporeactive to antibodies, and homogeneous, placing separate tissue specimens in each of a plurality of the compartments, adding to the internal volume of the container a medium that is in a liquid-state such that the divider and the specimen are at least partially immersed in the medium, allowing the medium to solidify to create a unitary block, and slicing the block along a plane that intersects the specimens, the divider material, and the medium, to create a thin section of the block that comprises the embedded specimens, the embedded divider material, and the medium. The section is adapted to be observed using a microscope or other observation equipment.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 is a flowchart of a prior art histology process, including a common formalin fixing and paraffin embedding (FFPE) step within the process.

[0017] Figures 2A-2C illustrate a divider tool in the form of a crossed-strip divider.

[0018] Figure 3 is a flowchart of steps of a method of the present disclosure.

[0019] Figure 4 schematically illustrates a thin section of a block, where the section includes a six-compartment divider, six tissue specimens (one per compartment), and a marker.

[0020] Figures 5A and 5B are microscope photographs showing two H&E stained dividers.

[0021] Figures 6A and 6B show two alternative divider designs.

[0022] Figure 7 schematically illustrates a method for producing a cup-shaped divider.

[0023] Figure 8 A is a top perspective view of a divider that comprises cups, made by the process illustrated in fig. 7.

[0024] Figure 8B illustrates a section of a paraffin block with embedded tissues created using the divider of figure 8A.

[0025] Figures 9A and 9B are top and front views, respectively, of a tool that comprises a porous bag with a plurality of separate compartments.

DETAILED DESCRIPTION

[0026] An embodiment of the system, tool and method of this disclosure includes the use of silk as the material for the tissue dividers and markers. Silk has been used in tissue engineering due to its mechanical strength and its inert chemical properties. Silk is also hypoimmunogenic and heat stable. The type of silk which is commonly used for bioengineering purposes is from the larva or caterpillar of the domesticated silk moth, Bombyx mori. The structure of silk is simple and homogenous in which the core fibroin fibers are coated and held by glue-like sericin proteins. The uniform beta-sheet structure of fibroin makes silk fibers strong, yet flexible. The simple component makes silk hyporeactive to antibodies and other detection reagents used in histopathology. Because silk is comprised of proteins, it behaves similarly to tissue in the way it adheres to glass slides, helping it to maintain its position while being stained. Although these characteristics of silk make the material ideal for this purpose, other materials with similar characteristics may also function as described herein, including, but not limited to spider silk, bamboo fiber, paper, cotton, and other natural or synthetic polymers. Divider materials comprising natural fibers or filaments are generally preferred.

[0027] The tissue divider leaves a faint boundary on the sections and the stained slides. The boundary provides a visual contrast from the paraffin in which it is immersed. Silk and paper based dividers are differentially stained by H&E where silk is stained but paper is not. This also gives researchers some flexibility of how well the boundary needs to be shown by using different dividers. This boundary can be used as a guideline to identify each individual tissue sample within the slide and scanned images. An automated identifying and analysis of the tissue samples from a digitized slide image may be employed, utilizing the boundaries of the tissue dividers, enabling counting of the number of nuclei (e.g., stained with Ki67) to access the proliferation rate, for example.

[0028] The embedding step of the FFPE process includes a container, which may be a metal base. The container has an interior volume. The container may contain paraffin that is heated to at least its melting point so as to keep the paraffin in a liquid state. A tool, in the form of a divider, is placed within the container to create a plurality of sub- volumes or compartments in the container. Processed tissue samples are then placed within the sub-volumes or compartments created by the divider. The container can then be further filled (as needed) with additional liquid paraffin. A top or histology cassette can be placed in the container so as to cover the tissues. The cassette can be placed in the liquid paraffin after the tissues and either before or after the additional paraffin is added. The container with the top and the immersed tissues is then placed at a lower temperature location so that the paraffin solidifies. Once the paraffin sets, the tissue samples are held together in the solid paraffin as a unitary block. The several sub-volumes or compartments are separated by the divider and so are isolated and distinct from one another. This allows individual or parallel processing of the tissue samples which may then remain together for all subsequent steps.

[0029] In an optional step, the divider can be asymmetric, so that the several compartments within the paraffin block have different shapes. This allows each individual tissue sample embedded within a paraffin compartment to be tracked during the sectioning, staining and the following processing/observation steps. Another optional step is to use a piece of divider material as a marker, placed in one or more particular locations in the paraffin before it is hardened, for example in one compartment. The markers may provide uniqueness, one compartment from another, or for one or each individual tissue sample within a unit. For example, the marker may be placed in a corner of a partitioned sample sub-volume or it may be placed between samples within the paraffin, according to the researcher's preference.

[0030] A marker or an asymmetrical piece of divider material may also be used in order to identify the orientation of a tissue sample. Some studies need to know the orientation of tissue in sections in order to correlate findings with other measurements. For example, a study of heart hypertrophy requires measurement of each chamber and muscle wall. These measurements are correlated with histology measurements using MRI ex-vivo imaging. One current method of measurement is obtained by first making a hole in the tissue sample being measured. The orientation of the sample may be determined by locating the hole to verify that the proper measurement is taken. By inserting a silk strip into one or two chambers of the heart, it is easy to identify the orientation of the tissue, because the strip stays with the tissue while being sectioned and stained. The silk strip may be inserted before or after ex-vivo MRI imaging because silk is MRI compatible.

[0031] The subject high-throughput histopathology tool allows paraffin-embedded tissue samples to be divided for biopsy/necropsy analysis. The unique mechanical, chemical, and molecular properties of silk are highly beneficial for the tool. Silk is cost effective. The divider not only reduces the time and labor in sectioning, staining and data analysis, but also reduces the use of materials, such as paraffin, cassettes, glass slides, chemicals, and reagents. Consequently, it reduces storage space for these materials. Also, the divider makes direct comparisons of tissues within groups possible. Multiple tissues from control and treated groups may be processed simultaneously, thus minimizing the error and deviation in the process. Further, use of the divider tool reduces the time needed to change slides to analyze multiple samples, therefore optimizing a pathologist's time. Still further, along with the tissues being analyzed, the tool adheres to slides and may be faintly stained to create a nice frame, improving the possibilities for automated identification and analysis of the tissues. Lastly, the tool may be used in other ways such as ex-vivo MRI imaging.

[0032] There are several basic designs of tissue dividers disclosed herein, although these designs are not limiting of the scope of divider shapes and arrangements that can be used.

Designs depicted herein include: crossed strips, cup-shaped, and teabag-like containers. Some of the drawings depict six (e.g., 2x3) compartments or sub-volumes as an example, but other numbers of compartments such as 2x4, 2x6, 3x3, 3x4, 4x12, etc., may be utilized, depending upon the number of samples desired. Similarly, the metal base container or mold may be

37x24x6 mm as an example, but other base molds, such as 24x24x6 mm, 30x24x6mm, etc., may be utilized, depending upon the size desired.

[0033] A crossed strip tool/divider design may be constructed by crossing thin strips of virgin silk from silk cocoons and tying the intersecting location with silk string to keep the intersection intact. This tool design can be used for larger tissue samples, while utilizing a minimal amount of material for the dividers. Other shapes can be created by tying the strips differently or altering the placements of the strips and/or tying or otherwise fixing them in different locations relative to one another.

[0034] A cup-shaped container/divider may be constructed by pressing divider material (e.g., silk) into complementary top and bottom molds. The divider material may be in the form of a sheet or it may be solubilized before molding or otherwise shaping the divider. The divider may be reshaped into cup-shaped compartments, creating as many compartments as can possibly fit in an area of interest. Such cup-shaped dividers provide a new method of creating a tissue microarray (TMA). Currently, a TMA is made by arranging tissue cores that are taken from individual paraffin units. By using cup-shaped dividers, small tissue biopsies that are pre- arranged within a single paraffin block can then be examined all together. Each tissue sample is placed in one cup-shaped compartment or sub-volume. Each resulting block sub- volume then ends up being framed by a ring of divider material after sectioning and staining. The divider material-framed isolation of each tissue sample provides advantages with regard to research automation because image analysis software can, among other things, utilize the grid created by the divider material ring frames for alignment and the tracking of specific samples. Tissue samples may be analyzed and identified with each of their positions within the ring frame. The dividers may be cup-shaped or square-shaped or any other desired shape. Having a visible frame around and between tissues has the advantage of possible process automation. Image analysis software, such as NIH ImageJ, may be used to overlay a grid aligning with the ring frame. Then each tissue may be identified by its position in the grid, relative to the other tissue samples. The software may also provide analysis of staining intensity or the number of particles that are being stained. Such analyses are similar to those used to analyze microarrays of DNA.

[0035] From a manufacturing point of view, it may be easier to shape a divider material by dissolving it into aqueous form first. Silk cocoons, for example, are separated with the use of a heated alkaline solution in order to dissolve sericin which holds the cocoons together. After a water rinse, the silk is dissolved in 9M lithium bromide. After dialyzing out lithium bromide, silk proteins are left in an aqueous form after the lithium bromide is removed, and the silk is ready to be formed and shaped as cups or as other desired container/divider shapes. These shapes are then used to hold tissue samples embedded within paraffin. Very small samples, such as needle biopsies or tumors from experimental animals, can be arrayed into cups and sectioned as a group. Thus, a cup-shaped divider is an effective method of creating a TMA for use with biopsies. This embodiment of the divider may also be utilized with tissue cores taken from paraffin units. All the tissue samples can be stained and analyzed at once.

[0036] Another container/divider is a bag-like structure with porous walls (similar to a teabag). The bag can have multiple separate compartments or slots. Tissues may be placed into each slot. If needed, the bag can then be sealed by a clip of some sort or another means. The divider material is porous and therefore able to serve as a tissue supporter for fixation in formalin and additional processing. In the tissue-embedding step of processing, the bag may be trimmed in order to fit into a base mold, typically with the slots aligned in the mold. After the tissue samples have been solidified in the paraffin block, the porous bag and the encapsulated tissues may be sectioned together. This teabag-like divider is designed for use with very small samples, such as needle biopsies, and delicate tissues. Since the tissues are carried inside the bag, transferring of tissues in processing and embedding is minimized. An advantage of a teabag-like divider is that it minimizes tissue handling between fixation, dehydration and embedding, thus minimizing tissue disruption and loss. This embodiment of the tool can also be used in fixation and dehydration steps because of the inert properties of the divider material.

[0037] The high-throughput histopathology system, tool and methods described herein may be utilized in many different research processing areas including, but not limited to:

Hematoxylin and eosin (H&E) pathology

Sirius red staining for fibrosis, image analysis

Analysis of protein expression/IHC

Analysis of RNA expression/ISH

Needle biopsy/direct tissue microarray (TMA)

Tumor heterogenicity analysis

Automated single-cell picking and sequencing/microarray

Silk pin marking used in ex- vivo MRI imaging

[0038] Figures 2A - 2C illustrate stages of creation of an embodiment of tool/divider 36 that comprises crossed strips. Untreated silk cocoons are cut into strips 30, 32, and 38 with desired varied lengths. Strips 30 and 32 have interfitting slots 31 and 33 as depicted in partial assembly 28, fig. 2A. The interfitted strips may be tied together with silk string 34 to create assembly 28a, fig. 2B. The width of the strips is limited by the depth of whatever container is used to hold completed divider 36. This particular configuration provides a 2 x 3 array divider or tool 36, creating six individual areas or compartments in which up to six separate tissue samples may reside.

[0039] Figure 3 is a flow chart 40 of steps in the present method that differ from flowchart 10, fig. 1. Flowchart 40 essentially replaces step 14 of fig. 1. The normal metal mold is used. The mold is partially filled with liquid paraffin, step 41. A divider is placed in the paraffin, step 42. The divider divides the interior volume of the mold into two or more separate sub-volumes, which are used as compartments for different tissue samples. Separate tissue samples are placed into one or more of the compartments, step 43. Additional liquid paraffin is added as needed to embed the divider and tissue samples, step 44. The mold is then cooled to allow the paraffin to harden, step 45, to create a unified block with an embedded divider and embedded tissues.

[0040] Fig. 4 is a top view of a thin section 50 of a paraffin block or unit with divider 52 formed as per fig. 2C, with tissue samples 1 -6 in the six compartments. Also shown is a marker 54 located in compartment 53. Section 50 can be created using a microtome that is used to slice the paraffin block (with the divider tool and the specimens) along a plane that intersects the specimen, the divider material, and the paraffin to create a thin section of the block that is adapted to be observed using a microscope or other observation equipment. Marker 54 identifies one particular compartment, which allows the researcher to maintain identification of the six tissue samples throughout processing and analysis of the section. Also, since marker 54 is asymmetric, if section 50 is rotated from the orientation shown in fig. 4 on any axis, marker 54 will appear different. Accordingly, if section 50 is rotated or even turned upside-down, it will be obvious to the user who will know to reorient the section so that the tissue samples are properly oriented. Note that the marker can have any desired shape. For example, it may be a small cylinder or pin and can be placed in one of the compartments, so each tissue sample can be identified by its position relative to the marker.

[0041] Figs. 5 A and 5B are photographs taken through a microscope, of a generally linear silk-based divider (at about 200x magnification) and a generally linear paper-based divider (at about lOOx magnification), respectively. The dividers were embedded in paraffin, sectioned with a microtome, and H&E stained. The photographs illustrate that the divider is visible and thus can act as a visual separator when tissue samples are observed by a person or a machine.

[0042] Figures 6A and 6B depict dividers/tools 70 and 90 that are two of almost unlimited possible additional tool/divider configurations. Tool 70 uses four ovoid-shaped dividers 71-74 that create eight separate compartments comprising internal compartments 75-78 and external compartments 79-82. The compartments are separated from one another by at least one portion of divider material. Tool 90 comprises arc-shaped dividers 91-94 that create nine compartments 95-103. The compartments are separated from one another by at least one portion of divider material. Tissue and/or a marker may reside in each compartment created by any of the possible divider wall and tool configurations that can be created according to this disclosure. There is essentially no limit to the number of possible divider shapes and compartment types and shapes that can be created in a tool.

[0043] Figure 7 depicts a method of producing a cup-shaped multi-compartment divider 140 (fig. 8 A) of a tool using silk or a similar material in the form of sheet 130. Upper mold 122 with protruding portions 124 is lowered onto lower mold 126 with equal and opposing cavities 128. The molds are pressed toward each other as depicted by arrow 132, forcing sheet 130 to assume and maintain a configuration established by the shapes 124 and 128. This same method of tool- forming may also be done with silk or another material in an aqueous form being set between the upper and lower molds to assume their shape, or the divider may be formed or created in other manners that would be apparent to one skilled in the art.

[0044] Figure 8 A shows tool 140 produced using the method of fig. 7, with six separate cup- shaped compartments 142. Figure 8B illustrates a section 144 after tool 140 has been embedded in paraffin, with samples Γ - 6' placed into the six cups, and then hardened and sectioned. Each specimen is surrounded by a ring of stained divider material 146, which serves to separate and delineate the six specimens.

[0045] Note that the quantity, size, shape, and arrangement of the compartments created using the divider/tool is open ended, limited only by physical constraints such as the size and shape of the mold, the divider material, and the sizes of the specimens.

[0046] Figures 9A and 9B illustrate a tool 150 that comprises a porous bag divided into five separate compartments. Bag 150 can be made of silk, paper, or other materials described herein. One possible configuration of a bag is shown, but as with the other tools the quantity, size, shape, and configurations of the separate compartments can be varied, as desired. In this non- limiting example, bag 150 is created using front wall 171, back wall 173, end walls 170 and 172, and bottom 178, along with internal dividing walls 174-177, to create five aligned open-top volumes 151-155. The walls are porous to allow for paraffin embedding. After tool 150 is placed in paraffin, a sample and/or a marker can be placed into any one or all of the five compartments. Alternatively, tool 150 can also be used as a means to allow handling of small or delicate samples during the steps that precede paraffin embedding. A clip or another means (not shown) can be used to close the open top of the bag if necessary (e.g., to close the bag so it can be used to handle the tissue samples during processing, and/or to shorten the bag so the mold does not need to be as deep as the height of the bag).

[0047] A number of implementations have been described. Nevertheless, it will be understood that additional modifications may be made without departing from the scope of the inventive concepts described herein, and, accordingly, other embodiments are within the scope of the following claims.

Claims

1. A method of preparing a histopathology sample using a container with an internal volume, comprising the steps of:

placing a divider in the container so as to divide the internal volume into separate compartments;

placing a tissue specimen in at least one of the compartments;

adding to the internal volume of the container, a medium that is in a liquid-state such that the divider material and the specimen are at least partially immersed in the medium;

allowing the medium to solidify, to create a block; and

slicing the block along a plane that intersects the specimen, the divider material, and the medium to create a thin section of the block that comprises the specimen, the divider material, and the medium, and that is adapted to be observed using a microscope or other observation equipment.

2. The method of claim 1 , wherein the divider adheres to glass.

3. The method of claim 1 , wherein the divider is non-magnetic.

4. The method of claim 1, wherein the divider is resistant to ethanol and xylene.

5. The method of claim 1, wherein the divider is free of RNA and DNA.

6. The method of claim 1, wherein the divider is hyporeactive to antibodies.

7. The method of claim 1, wherein the divider is homogeneous.

8. The method of claim 1, wherein the divider comprises silk.

9. The method of claim 1 , wherein the divider comprises paper.

10. The method of claim 1, wherein the divider comprises spider silk.

11. The method of claim 1 , wherein the divider comprises cotton.

12. The method of claim 1, wherein the divider comprises a synthetic polymer.

13. The method of claim 1, wherein the divider comprises an asymmetrical pattern, so that the compartments are asymmetrical.

14. The method of claim 1 , wherein the divider comprises a plurality of separate cup-shaped internal volumes.

15. The method of claim 14, wherein the cup-shaped internal volumes are formed.

16. The method of claim 1 , further comprising placing a marker in a compartment.

17. The method of claim 16, wherein the marker is asymmetrical.

18. The method of claim 1 , wherein the divider comprises a porous bag.

19. The method of claim 18, wherein the porous bag comprises a plurality of separate compartments.

20. A method of preparing a histopathology sample using a container with an internal volume, comprising the steps of:

placing a divider in the container so as to divide the internal volume into a plurality of separate compartments, wherein the divider comprises silk and adheres to glass, is non-magnetic, resistant to ethanol and xylene, free of RNA and DNA, hyporeactive to antibodies, and homogeneous;

placing separate tissue specimens in each of a plurality of the compartments;

adding to the internal volume of the container, a medium that is in a liquid-state such that the divider and the specimen are at least partially immersed in the medium;

allowing the medium to solidify, to create a block; and

slicing the block along a plane that intersects the specimens, the divider material, and the medium to create a thin section of the block that comprises the specimens, the divider material, and the medium, and that is adapted to be observed using a microscope or other observation equipment.