[0001] The present invention is in the field of tissue sample analyses, and to the particular field of obtaining, handling and processing tissue biopsy samples.

BACKGROUND OF THE INVENTION

[0002] Diagnosis of disease often requires microscopic examination of tissue biopsies. To minimize the risk of the biopsy, the amount of tissue taken is the minimum amount generally considered sufficient for an accurate diagnosis. For example, breast and prostate biopsies are commonly performed by inserting small needles into these organs, and drawing small amounts of tissue into them. Such a procedure is known as a "needle core biopsy" (NCB).

[0003] The length of most breast and prostate NCBs is generally between one half and one inch (approximately 12 to 25 millimeters), and their diameter is generally between twenty-five and seventy-five thousandths of an inch (approximately 0.5 to 2.0 millimeters). A typical prostate NCB is about 15 millimeters long, and about 1 millimeter in diameter.

[0004] Although NCBs are intended to be precise procedures, actual evidence of disease {e.g. cancerous cells) may be present in only a small portion of the biopsy. This may happen because the lesion itself is small, or because the needle passed through only a small part of the lesion. As a result, for accurate diagnosis, it is essential that as much of the needle core biopsy be made available for examination as possible.

[0005] Following removal of the tissue sample from the patient, NCBs are generally treated according to the following multi-step protocol.

[0006] First, fixation involves removing the NCB from the needle (or similar such harvesting device), and then placing in a solution, usually 10% neutral buffered formalin, which denatures enzymes in the tissue and thereby stops all metabolic processes. This is intended to prevent the tissue from decomposing before it can be examined.

[0007] Second, the needle core biopsy is processed so that it can be cut into thin sections. This processing generally involves immersing tissues first in alcohol, then in xylene, and finally in liquid paraffin. During processing, the water inside the cells of the tissue is replaced by progressively less hydrophilic liquids, the last of which is paraffin.

[0008] Tissues cannot be cut by a microtome or similar device into thin sections in their native state, .Le. they are not "microtome sectionable" as this term has been used in the art. However, when the water in the tissue is replaced by liquid paraffin during processing, and the tissue is then cooled to a temperature at which the paraffin solidifies, the tissue can then be cut into thin sections, and is therefore "microtome sectionable".

[0009] Third, the fixed, processed NCB is embedded by. placing it in warm, liquid paraffin, and then permitting it to cool and form a solid rectangular block of paraffin containing the fixed, processed, NCB.

[0010] Fourth, the paraffin block containing the fixed, processed NCB is sectioned (Le. cut into thin strips) by a microtome or similar such device. Those strips that contain fragments of the NCB are placed on a glass slide. To obtain the uniform thickness of the very thin sections that is required for tissue analysis, the density of the material surrounding the tissue should be as close as possible to the density of the processed tissue. Ideally, both the .tissue and the material surrounding should be composed primarily of the same material, e.g. paraffin.

[0011] Fifth, the material on the glass slide is stained with various pigments that make the structure of the tissue, and the individual cells of which it is composed, easier to see.

[0012] Sixth, and finally, the stained tissue on the glass slide is examined by a physician, who makes a diagnosis based, at least in part, on the appearance of the tissue that is available for review. [0013] In the third and fourth steps above, vitζ. embedding and sectioning, there is a problem with the existing technology. More specifically, when a tissue sample is held or supported by a conventional tissue biopsy support, uniform sections of the NCB are difficult to obtain. This is because convention tissue biopsy supports are made of materials other than paraffin, such as fluoropolymers, which have densities and/or hardnesses different from the tissue and/ or the paraffin. These materials present a different resistance to the cutting blade than the paraffin or the tissue, which causes imperfect sectioning.

[0014] In addition, when materials with densities or other properties different from paraffin surround a tissue in a paraffin block, there is an increased chance that the cut slice will disintegrate. That is, the tissue may separate from these other materials when or after the paraffin block is sectioned, or the combination of these other materials and the tissue will separate from the paraffin when or after the paraffin block is sectioned.

[0015] Finally, when materials other than paraffin surround a tissue in a paraffin block, there is an increased chance that the exchange of tissue water for paraffin will be mechanically blocked, making the tissue more difficult to subsequently section.

[0016] There is therefore a need in the art for a scaffold that permits a tissue sample to be supported and oriented by the same material used for embedding, e.g. paraffin.

[0017] Moreover, in the third step above (i.e. the tissue is embedded), there is an additional problem with the current art — the orientation of the NCB in the paraffin block determines what percentage of the cross-sectional area of the tissue will appear on an individual slide.

[0018] In a paper titled "Individual prostate biopsy core embedding facilitates maximal tissue representation" by Jerry Kao, Melissa Upton, Ping Zhang, and Seymour Rosen, published in the Journal of Urology in 2002, Volume 168, pages 496-499, the authors used a computer simulation to compute the consequences of even small deviations from ideal orientation of the needle core biopsy in the paraffin block. Their findings were as follows.

[0019] First, if a simulated needle core biopsy, 15 millimeters in length and 1 millimeter in diameter, was oriented in the paraffin block exactly on the plane of the microtome blade, the maximum surface area of the biopsy available for examination on a single slide would be 15 mm ² , or 100% of the simulated needle core biopsy's maximum cross-sectional area.

[0020] Second, if the simulated needle core biopsy was oriented only 3 degrees away from the plane of the microtome blade, the maximum surface area of the biopsy available for examination on a single slide would be 13.3 mm ² , or 87% of the simulated needle core biopsy's maximum cross- sectional area.

[0021] Third, if the simulated needle core biopsy was oriented only 5 degrees away from the plane of the microtome blade, the maximum surface area of the biopsy available for examination on a single slide would be 9.01 mm ² , or 60% of the simulated needle core biopsy's maximum cross- sectional area.

[0022] Fourth, if the simulated needle core biopsy was oriented only 10 degrees away from the plane of the microtome blade, the maximum surface area of the biopsy available for examination on a single slide would be 4.52 mm ² , or 30% of the simulated needle core biopsy's maximum cross- sectional area.

[0023] These findings demonstrate the need for a scaffold that properly orients breast and prostate needle core biopsies in paraffin blocks as part of the process of preparing these tissues for subsequent examination by a physician.

[0024] Both the need for a more uniform embedding medium and the need for a method to insure optimal orientation are not confined to breast or prostate biopsies, or just to biopsies obtained with a hollow-bore needle, or even to tissues embedded in paraffin, for the following reasons:

[0025] 1) Breast and prostate biopsies comprise only about half of all biopsies performed. There are approximately as many skin biopsies performed as breast biopsies each year. The number of muscle, nerve, liver, kidney, intestine, testis, cervix, uterus, lung, heart, and even brain biopsies performed each year approximates the number of skin, breast, or prostate biopsies performed each year. The same considerations about orientation of breast and prostate tissues apply to these tissues as well. In addition, skin biopsies must be oriented so that a full-thickness cross-section of the tissue appears on each slide, because diseases of the skin are often diagnosed by the presence of disease in one layer of skin and the absence of disease in another layer.

[0026] 2) While some biopsies are obtained with hollow-bore needles, other biopsies (such as skin) are obtained with punch needles and sώl others are obtained by cutting wedges of a tissue with a knife or similar device. Orientation is equally important regardless of the method by which the tissue is obtained.

[0027] 3) While paraffin is the most common embedding agent in current use, it is not the only such agent For example, methacrylates are used in preparing tissues for electron microscopy examinations. AU these embedding agents work in the same way, vitζ. they replace water in the tissues, thereby making the tissues sectionable in a microtome.

[0028] Many devices have been used for many years to achieve better orientation of tissues during the embedding process. Three examples of such devices are described below

[0029] First, Robert J. Swan and Hugh W. Davis published "The Biopsy-Cucumber Unit. A Method to Improve Tissue Orientation" in 1970 in Obstetrics and Gynecology, Volume 36, Number 5, pages 803-805. The authors begin their paper by noting that "[a]ccurate histologic diagnosis of early cervical neoplasia is often hampered by the poor orientation of tissue biopsies" and propose the use of certain materials to support and orient a tissue sample for sectioning. Among the suitable materials, the authors note "gelfoam radish, potato, and cucumber slices" could be employed as "substances upon which biopsies could be mounted and maintained so that technicians could consistently section biopsies perpendicular to the epithelial surface . . ." Particular preferred as the tissue sample support material was cucumber slices, as "there is no interference with the staining procedure. The unit is oriented in the liquid paraffin so that when the block is mounted, the microtome will cut perpendicular to the biopsy. Orientation is preserved . . . thus eliminating one more stumbling block toward the more accurate diagnosis of available tissue." One drawback to the cucumber slices, however, was that they require a substantial dehydration process prior to use as a tissue sample support and orientation material.

[0030] Second, W. R. Chatfield and A. D. Bremner described an "Intrauterine Sponge Biopsy. A New Technique to Screen for Early Intrauterine Malignancy" in a paper published in 1972, also in Obstetrics and Gynecology, Volume 39, Number 2, pages 323-328. According to their proposal, "[a]n abrasive polyvinyl sponge is passed into the uterus in an [intrauterine contraceptive device] introducer, and as it is withdrawn from the uterus, it abrades and absorbs the tissue. The entire sponge is processed as a routine histologic specimen."

[0031] Third, U.S. Patent No. 5,817,032 and U.S. Patent No. 7,156,814 describe and claim "a microtome sectionable tissue support" which is formed of "material which can be successfully sectioned in a microtome, is resistant to histological stains, and is resistant to degradation from solvents and chemicals used to fix, process, and stain tissue" along with a suitable embedding material. These patents disclose polymers, particularly fluoropolymers, as the material for use in these devices.

[0032] A problem encountered with each of these devices is that they are all formed of materials which differ in density and/ or hardness from paraffin and/or tissue. As a result, the use of these devices frequently suffer from the problems noted above, vi _ζ , (1) it is difficult to obtain uniform slices because of the difference in hardness and/ or density between the tissue support device and the paraffin and/or the tissue; (2) the material comprising the tissue support device may not adhere adequately to the tissue sample and/ or to the paraffin, or even to the slide on which the sample is to be examined; and (3) the tissue support device can interfere with the interaction between the paraffin and the tissue sample, including inhibition of the replacement of water or other fluid in the tissue with paraffin (this is particular true of the devices in the '032 and '814 patents, which are designed to be interposed between the tissue sample and the embedding paraffin).

[0033] Paraffin, however, cannot itself be used as the material for a tissue orientation device because paraffin is a brittle solid at room temperature.

[0034] There is therefore a need in the art for a device for orienting a tissue sample that does not suffer from one or more problems of the existing art. There is also a need for a device that not only properly orients tissue in a paraffin block, but also reduces the risk that tissue in the paraffin block may be lost during sectioning the block and/or transferring thin sections to a glass slide for examination. There is still also a need for a device that does not potentially obstruct the flow of fixatives into and out of the tissue during processing.

SUMMARY OF THE INVENTION

[0035] Accordingly it is an object of the present invention to provide a scaffold that supports and orients a tissue sample for processing and analysis ^" .

[0036] It is another object of the present invention to provide a scaffold that can maintain the preferred orientation of the tissue sample from the time of initial gross-in throughout the tissue processing procedure and continuing through the wax embedding stage with no human involvement required beyond initial gross-in. [0037] It is another object of the present invention to provide a scaffold for efficiently harvesting tissue samples for biopsy.

[0038] It is another object of the present invention to provide a method for handling harvested tissue samples in an efficient manner with a minimum of human intervention.

[0039] It is another object of the present invention to provide a scaffold that can retain tissue samples and facilitate easy transfer of the specimen without having to individually retrieve small tissue fragments from a sample container.

[0040] It is another object of the present invention to provide a scaffold that can be inserted into a tissue cassette, mesh bag, lens paper, sponge or other mateάal(s) during the processing of the immobilized tissue.

[0041] It is another object of the present invention to provide a scaffold which assures that the tissue will be oriented in the desired sectioning plane.

[0042] Still other objects of the present invention are directed to methods of using the foregoing tissue sample support and orientation devices.

[0043] In accordance with this and other objects, a first embodiment of the present invention is directed a scaffold for orienting a tissue sample comprising at least one hydrogel or organogel or hydrocolloid, which contains at least one component that is substantially liquid in the temperature range of 4°C to 37 ⁰ C.

[0044] The inventive scaffold is: (a) sufficiendy flexible in the temperature range to permit the scaffold to be to be bent (or otherwise manipulated) and yet then return to- substantially the same shape; (b) sufficiently rigid in the temperature range to maintain a tissue sample in a particular pre-determined orientation; and (c) not microtome sectionable. [0045] The component which is substantially liquid in the inventive scaffold can be replaced during fixing and processing of said tissue with one or more components that are substantially solid in the temperature range. The "scaffold" and tissue sample are then microtome sectionable.

[0046] Another embodiment of the present invention is directed to a method of processing tissue for examination using a scaffold of the present invention.

[0047] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are intended to provide further explanation of the invention as claimed.

BRIEF DESCRIPTION OF THE FIGURES

[0048] FIG. 1 is a front view of a first embodiment of a scaffold of the present invention.

[0049] FIG. 2 is a side view of the embodiment illustrated in FIG. 1.

[0050] FIG 3 is a top view of the embodiment of the scaffold illustrated in Figures. 1 and 2.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0051] The present invention will be described in more detail with reference to the embodiments shown in the attached drawings.

[0052] Referring to the drawings, Figures 1, 2 and 3 show a scaffold for tissue sample orientation 10 according to a first embodiment of the present invention. Broadly, this scaffold for tissue sample orientation comprises: (i) a generally flat base member 12; and (ii) a plurality of support members 14 arranged on the base member 12 in a predetermined spaced relationship.

[0053] According to certain preferred embodiments, the support members 14 may be arranged in substantially parallel rows on the base member 12. According to alternative, but equally preferred, embodiments, the support members may be arranged in staggered rows on base member 12. The base member 12 may be of any suitable dimensions for the intended use thereof. For example, when the inventive scaffold for tissue sample orientation is to be used in conjunction with a standard tissue cassette, the inventive scaffold for tissue sample orientation is preferably dimensioned to fit within such a standard tissue cassette without any bending, folding or cutting. Illustrative values for the dimensions of base member 12 in such an embodiment are about 25 mm x 25 mm x 1 mm. The base member 12 also preferably includes a plurality of holes and/or slits dimensioned to permit liquid to pass through.

[0054] The rows of support members are generally spaced apart a suitable distance for their intended purpose. That is, the rows of support members are spaced apart a sufficient distance to allow a tissue sample to be placed between two adjacent rows and to maintain that tissue sample in the same orientation during further processing, while not adversely affecting the tissue sample. In any of the embodiments of the present invention, the support members within any particular row may be a uniform or a varying distance from one another. Preferably, at least a plurality of support members within a particular row are a uniform distance from one another.

- [0055] In practice, the rows of support members are preferably spaced between 1.0 and 2.5 mm apart, e.g. 1.2 mm, 1.6 mm, 1.8 mm or 2.0 mm apart. Within a given row, the support members are preferably spaced between 0.1 and 1.0 mm apart, e.g. 0.2 mm, 0.3 mm or 0.5 mm apart. One skilled in the art may determine the particular dimensions of the spaced relationship of the support members empirically, based, for example, on the size, or sizes, of the tissue sample(s) to be examined.

[0056] Referring to FIG. 3, a preferred exemplary arrangement of a plurality of support members 14 on a base member 12 is illustrated. According to this embodiment, the support members 14 are arranged in a plurality of substantially parallel rows along the directions of both the x-axis and the y-axis of the base member 12. According to this illustrative embodiment, the base member 12 may be about 25 mm x 25 mm and the parallel rows of support members 14 are separated along the x-axis by two possible spacing arrangements — one spacing being sufficient to receive a tissue sample from a 14 gauge needle (100) and the other spacing being sufficient to receive a tissue sample from a 16 gauge needle (200). In addition, a third spacing is provided along the y- axis (300), this one being sufficient to receive a tissue sample from an 18 gauge needle. According to certain preferred embodiments of this exemplary arrangement, a single, continuous row of support members is provided around the perimeter of the base of the scaffold for tissue sample orientation to facilitate retention of the tissue sample(s) during subsequent processing.

[0057] Each of the support members 14 has a stem portion 16 projecting substantially upright from the base 12, and a head portion 18 formed at a distal end of the stem portion 16. The head portion 18 is dimensioned to engage and retain a tissue sample during processing. The stem portion 16 has a proximal end 22 connected to the major surface 20 of the base 12, and a distal end 24 connected to the head portion 18.

[0058] As noted above, the scaffold for tissue sample orientation according to the present invention may have any suitable dimensions and/or shape. For example, in the scaffold for tissue sample orientation 10, the base member 12 may be formed in any suitable dimension and shape that can firmly support the support members 14, such as a rectangular, circular, or elliptical shape. Preferably, base member 12 is approximately square in shape. The thickness of the base member 12 is preferably in a range of 0.5 mm through 5.0 mm, although this may be varied as desired depending upon the particular use of the inventive device. According to particularly preferred embodiments, the inventive scaffold for tissue sample orientation is dimensioned such that it can fit within a tissue cassette for further processing.

[0059] The stem portion 16 of the support member 14 may have various shapes, such as a cylindrical, prism or frustoconical shape, and more than one stem portion may be provided for each head portion 18. Further, a radiused corner having a predetermined radius of curvature may be provided to a junction area between the proximal end 22 of the stem portion 16 and the major surface 20 of the base member 12, for attenuating a stress concentration caused by the deflection of a support member 14. The stem portion 16 is preferably between 0.1 and 1.0 mm in height {i.e. from the base member 12 to the bottom of head portion 18) and the head portion 18 between 0.1 and 0.5 mm high when viewed from the side as in Figures 1 and 2.

[0060J According to certain preferred embodiments of the present invention, the stem portion 16 is a cylindrical shape having a diameter preferably between 0.1 and 1.0 mm. In such embodiments, the stem portion 16 may have a uniform diameter along its entire length or the diameter may vary. According to certain preferred embodiments, the diameter of the stem portion 16 varies along its length to form one or more grooves to facilitate the positioning of a tissue sample.

[0061] The head portion 18 may have various shapes besides the hemispherical shape illustrated in Figures 1 and 2. Illustrative examples of suitable shapes include, but are not limited to, shapes such as hook, conical, cylindrical, spherical, pyramidal and hemispherical (umbrella) shapes. Asymmetrical shaped head portions 18 {e.g. cones, pyramids and hemispheres) may be formed in any orientation on the stem portion 16, e.g. if the head portion 18 is a conical shape, the stem portion 16 may be joined to the apex of the cone, the base of the cone or the side of the cone. The size of the head portion 18 must be sufficient to retain a tissue sample when placed in the inventive tissue sample support and orientation device. According to certain preferred embodiments, when viewed from above (as in Figure 3), the head portion 18 is preferably not more than 0.2 mm in any direction.

[0062] According to certain preferred embodiments, for a support member 14 having a cylindrical stem portion 16 and a hemispherical head portion 18, the minimum diameter of the stem portion 16 is preferably in a range of 20% to 60% of the maximum diameter of the head portion 18, in order to obtain sufficient engagement-retaining force on the tissue sample. Also, the peripheral edge of the head portion 18 is preferably formed with no sharp-edges, for reducing possible damage to the tissue sample when the head portion is engaged therewith.

[0063] The scaffold for tissue sample orientation according to the present invention may be made of any suitable material capable of imparting the desired characteristics to the inventive scaffold. Particularly suitable materials are hydrogels, organogels and hydrocolloids.

[0064] As used herein, a "hydrogel" is intended to mean a network of water-insoluble natural or synthetic polymer chains dispersed in an aqueous medium, such as water. Illustartive examples of synthetic polymers which may be found in a hydrogel include, but are not limited to, silicones, polyacrylamides, polyethyelene oxides, polyvinylpyrrolidones, polyvinyl alcohols and acrylates. Illustrative examples of natural polymers include, but are not limited to, agarose, cellulose, methylcellulose and hylaronan. Other suitable hydrogels are known and commercially available.

[0065] As used herein, an "organogel" is intended to mean a non-crystalline, non-glassy thermoreversible solid material composed of a liquid organic phase, such as an organic solvent, mineral oil or vegetable oil entrapped in a structuring network, such as lecithin. Suitable organogels are known and commercially available.

[0066] As used herein, a "hydrocolloid" is intended to mean a colloid system wherein the colloid particles are dispersed in water. A hydrocolloid has colloid particles spread throughout water and, depending on the quantity of water available, can take on different states, including gel or sol (liquid). Hydocolloids can be irreversible (single state) or reversible, such as agar. Other illustrative examples of suitable hydrocolloids include, but are not limited to, carrageenan, gelatin and pectin. Still other suitable hydrocolloids are known and commercially available.

[0067] The base member 12 and the head portion 18 and the stem portion 16 of the support members are preferably made of the same material. The inventive scaffold is sufficiently flexible to permit the inventive scaffold for tissue sample orientation to be bent (or otherwise manipulated) so as to facilitate placement of the tissue sample in the device, and yet then return to substantially die same shape as it was before such bending or manipulation.

[0068] The scaffold for tissue sample orientationaccording to the present invention may be formed by various methods known and available to Λose skilled in the art. To easily form support members 14 having unique shapes, it is advantageous to integrally mold the base and the headed elements by an injection molding process using a destructible stem mold, as is disclosed, for example, in U.S. Patent No. 5,242,646, the contents of which is incorporated herein by reference.

[0069] The scaffold for tissue sample orientation of the present invention is particularly useful for processing biopsy tissue samples for analysis.

[0070] Broadly, the method of preparing biopsy tissue samples for histological examination comprises: (a) removing a tissue sample from a patient; (b) placing the tissue sample onto a scaffold of die present invention; (c) processing the tissue sample. Such processing includes subjecting both the inventive scaffold and the tissue sample immobilized thereon to a process for replacing tissue fluid with wax and diereby impregnating die tissue sample with wax, and dien embedding in a wax mold to form a solid block of wax. Subsequentiy, a microtome may then be used to slice the solid block of wax (including the tissue sample and inventive scaffold) into diin sections which can be used for further examination and analysis.

[0071] The foregoing description and the following examples are illustrative only and are not intended to limit the scope of the invention as defined by the appended claims. It will be apparent to those skilled in the art that various modifications and variations can be made in the methods of the present invention without departing from the spirit and scope of the invention. Thus, it is intended diat the present invention cover the modifications and variations of this invention provided diey come within die scope of die appended claims and their equivalents.