Source: http://www.google.com/patents/US7776274?dq=6514640
Timestamp: 2017-09-25 09:22:23
Document Index: 464165992

Matched Legal Cases: ['Application No. 06076973', 'Application No. 08013740', 'Application No. 02773621', 'Application No. 98951995', 'Application No. 2004', 'Application No. 2004']

Patent US7776274 - Cassette and embedding assembly for handling and holding tissue samples ... - Google Patents
The disclosure relates to a cassette, frame and mold for holding a tissue sample during an embedding and mircotoming process, and related methods. The cassette is sectionable in a microtome and includes a body with a bottom wall and a plurality of side walls. First and second side walls are generally...http://www.google.com/patents/US7776274?utm_source=gb-gplus-sharePatent US7776274 - Cassette and embedding assembly for handling and holding tissue samples during processing, embedding and microtome procedures, staging devices therefore, and methods therefor
Publication number US7776274 B2
Application number US 11/072,119
Also published as CA2499369A1, CA2499369C, DE60236764D1, EP1545775A1, EP1545775A4, EP1545775B1, EP2002894A1, EP2002894B1, US8609431, US9097630, US20050147538, US20100278627, US20140099661, WO2004028693A1
Publication number 072119, 11072119, US 7776274 B2, US 7776274B2, US-B2-7776274, US7776274 B2, US7776274B2
Inventors Warren P. Williamson, IV, Craig B. Berky, Stephen P. Whitlatch, Thomas J. Ward
Original Assignee Biopath Automation, L.L.C.
Patent Citations (155), Non-Patent Citations (27), Referenced by (12), Classifications (16), Legal Events (2)
US 7776274 B2
The disclosure relates to a cassette, frame and mold for holding a tissue sample during an embedding and mircotoming process, and related methods. The cassette is sectionable in a microtome and includes a body with a bottom wall and a plurality of side walls. First and second side walls are generally V-shaped to present an apex of the “V” to the microtome blade. A lid of the cassette is stiffer than the bottom wall of the cassette to assist with positioning the tissue sample. The side walls of the cassette are perforated so as to significantly reduce the amount of cassette material that must be cut by the microtome blade. In one embodiment, to additionally reduce blade wear the ribs on one side wall are offset lengthwise relative to the ribs on an opposite side wall. An upper flange of the cassette includes depressions configured to register with detents in the frame.
1. A cassette for holding a tissue sample, comprising:
a body including a perforated bottom wall and a plurality of perforated side walls extending upwardly with respect to said bottom wall to define an interior space for receiving the tissue sample;
said bottom wall and said plurality of side walls being constructed of a material capable of being successfully sectioned in a microtome and resistant to degradation during histologic tissue processing, wherein said plurality of side walls comprise first and second side walls each having a length and extending upwardly from opposite edges of said bottom wall, at least one of said first and second side walls including portions angling toward the other of the first and second side walls on the opposite edge of the bottom wall to define an outwardly projecting portion proximate a midpoint along said length of said at least one side wall.
2. The cassette of claim 1, further comprising a lid configured to be coupled with said body and movable between open and closed positions, said lid being stiffer than said bottom wall.
3. The cassette of claim 1, wherein said plurality of side walls comprise perforated walls in which the ratio of open area to solid area is at least about 3.5:1.
4. The cassette of claim 1, wherein said plurality of side walls comprise perforated walls in which the ratio of open area to solid area is at least about 3.0:1.
5. The cassette of claim 1, wherein said plurality of side walls comprise perforated walls in which the ratio of open area to solid area is at least about 2.5:1.
6. The cassette of claim 1, wherein said first and second side walls include a length and comprise perforated walls constructed with spaced apart ribs, said ribs of said first side wall being offset along said length with respect to said ribs of said second side wall.
7. The cassette of claim 1, further comprising:
a flange extending along upper portions of at least two of said side walls, said flange including depressions configured to register with detents in a frame during a tissue embedding process.
8. The cassette of claim 1, wherein said bottom wall includes a central area and comprises a perforated wall constructed with spaced apart ribs, said ribs angling toward said central area.
The present application is a continuation of PCT Serial No. PCT/US02/30775 filed on Sep. 26, 2002 (now pending), the disclosure of which is hereby fully incorporated by reference herein.
In spite of the various advances made in this field, there is an increasing need for additional improvements related to increased production capability and increased and more consistent quality of embedded tissue samples and resulting slices or ribbons of embedded tissue which will be subject to diagnosis.
Generally the invention relates to a cassette for holding a tissue sample during an embedding and microtoming or slicing process. The cassette includes a body with a bottom wall and a plurality of side walls extending upwardly with respect to the bottom wall to define an interior space for receiving the tissue sample. The bottom wall and the plurality of side walls are constructed of a material capable of being sectioned in a microtome. Preferably, the cassette material is also resistant to any type of degradation during processing which would compromise its ability to function in accordance with the invention. In a first aspect of the invention, the plurality of side walls comprise first and second side walls on opposite sides of the bottom wall each including portions angling from approximate midpoints of the first and second side walls toward the other of the first and second side walls on the opposite side of the bottom wall. In the preferred embodiment, the two longest side walls of the four side walls comprising a rectangular cassette are generally V-shaped in a direction away from the interior of the cassette. This presents an apex of the V-shape to the microtome blade, after the embedding process is complete, which assists with the cutting action. Specifically, this feature has been found to reduce or prevent the hardened paraffin from breaking or cracking away from the cassette side wall material while making slices in the microtome.
The cassette preferably further includes a lid configured to be coupled with the body for movement between open and closed positions. The lid may be depressed downwardly on top of the tissue sample in the cassette interior. The lid is preferably stiffer than the bottom wall of the cassette. This feature allows the lid to position the tissue sample in the cassette parallel to the bottom of the mold during the embedding process. More specifically, the stiffer lid pushes the tissue sample and the more flexible bottom wall of the cassette against the rigid bottom of a base mold while the molten paraffin solidifies. This helps ensure that the bottom wall of the cassette can be removed in its entirety during a facing operation in the microtome prior to slicing the tissue sample, and that the tissue is positioned flatly against the bottom wall of the cassette.
In another aspect of the invention, the side walls of the cassette are perforated such that there is at least a ratio of about 2.5:1 of open area to solid material area whereby the solidified paraffin occupies the open area of the side walls. This ratio may be altered by using different materials for the cassette and/or paraffin embedding media. For instance, a higher molecular weight paraffin or lower moneluculer weight cassette will allow the ratio to vary somewhat. Currently, an industry standard paraffin (e.g., Sakura VIP processing/embedding medium) works best with a ratio of at least about 3.0:1 and, more preferably, at least about 3.5:1. This reduces the amount of cassette material that must be cut by the microtome blade while taking slices of the tissue and, therefore, increases blade life and quality of the resulting ribboned, embedded tissue samples. In addition this ratio assures that the paraffin is strong enough not to fracture when cut by the microtome blade. Another feature that results in similar advantages involves forming the side walls from ribs and offsetting the ribs of one of the first and second side walls with respect to those of the opposite side wall along the side wall length. Thus, the microtome blade will contact a more uniform amount of the cassette material along its length during each pass. This significantly reduces blade wear while cutting through the cassette material. Reduced blade wear is advantageous to keeping blade costs under control as the majority of blades used are disposable.
In another aspect of the invention, the cassette further includes a flange extending along upper portions of at least two of the side walls. The flange includes depressions configured to register with detents in a frame during the tissue embedding process. This increases the effective height dimension of the cassette interior, thereby allowing more tissue to be placed in the cassette and more passes to be made in the microtome. In this regard each pass of the microtome may only take a 5 micron slice. Therefore, using depressions having a depth, for example, of 0.14″ can allow about 70 more slices to be taken in the microtome.
The invention further contemplates the various unique assemblies of two or more of the tissue cassette, frame, and base mold as disclosed herein. With respect to the frame and base mold, for example, structure is provided to physically hold the frame against the base mold. In the preferred embodiment, a seal is provided to perform this holding function and also to prevent leakage of liquid paraffin from the base mold.
In another aspect, the invention contemplates a device for staging a tissue sample cassette from an upper position in a frame used during a tissue embedding process to a lower position in the frame. The device includes a handle, a staging mechanism coupled with the handle and configured to engage an upper surface of the cassette, and a stop operatively coupled to the handle and configure to stop vertical movement of the staging mechanism when the cassette reaches the lower position in the frame. The staging mechanism further comprises a plurality of fingers which are configured to engage a corresponding plurality of locations on the upper surface of the cassette. For example, four fingers may be provided for engaging four corner locations on the cassette. This helps ensure that the cassette is engaged with at least four pairs of detents located proximate corner portions of the cassette thereby positioning the bottom wall of the cassette parallel to and against the bottom wall of the base mold.
In one embodiment, the staging device is a rigid member and the stop comprises a fixed stop member coupled for movement with the staging mechanism and configured to stop against an upper surface of the frame. In another embodiment, the device includes a stabilizing mechanism coupled with the handle and moveable relative to the staging mechanism. The stabilizing mechanism is configured to engage an upper surface of the frame as the staging mechanism moves the cassette from the upper position to the lower position within the frame. In this embodiment, the staging mechanism is normally spring-biased into an upward position and is forced downward against the spring bias when moving the cassette from the upper position to the lower position. The stop in this embodiment further comprises respective surfaces of the handle and stabilizing mechanism which engage one another when the staging mechanism has placed the cassette into the lower position. The staging devices of this invention ensure that the cassette is fully staged into the base mold, while ensuring that the cassette is not pushed too far through the frame. Moreover, the staging devices ensure that the bottom wall of the cassette and, therefore, the tissue sample, lay flat against the bottom of the base mold. This improves the efficiency and quality of tissue shavings later made in the microtome.
The invention also encompasses the various methods of using the tissue cassette and cassette/frame/base mold assemblies as disclosed herein.
FIG. 1 is an exploded perspective view of an assembly including the tissue cassette, the frame into which the cassette is inserted and the base mold into which the frame and cassette assembly are inserted.
FIG. 2 is an assembled perspective view of the tissue cassette, frame and base mold.
FIG. 3 is a cross sectional view taken along line 3-3 of FIG. 2 and showing the tissue cassette in its initial upper position.
FIG. 4 is an assembled perspective view, similar to FIG. 2 but illustrating the second, staged position of the tissue cassette.
FIG. 5 is a cross sectional view taken along line 5-5 of FIG. 4 and showing the tissue cassette staged down into its second, lower position against the bottom of the base mold.
FIG. 6 is a cross sectional view of the frame and the tissue cassette embedded in material such as paraffin, after release from the base mold.
FIG. 7 is a perspective view of a manual, spring-biased staging device.
FIG. 8 is a longitudinal cross sectional view of the staging device of FIG. 7 being used to stage a tissue cassette through the frame and into the base mold.
FIG. 9 is a perspective view of a tissue cassette constructed in accordance with a second embodiment of the invention.
FIG. 11 is a perspective view of a manual staging device constructed in accordance with a second embodiment.
FIG. 12 is a bottom perspective view of the staging mechanism associated with the staging device of FIG. 11.
Turning first to FIGS. 1-3, a tissue cassette 10 constructed in accordance with the invention is received within a frame 12 and the tissue cassette 10 and frame 12 are then positioned in a base mold 14. After a staging operation the base mold 14 is subsequently filled with liquid paraffin, as described further below. Tissue cassette 10 includes a perforated body 20 formed by four side walls 22 a, 22 b, 22 c, 22 d and a bottom wall 24. Each wall is preferably constructed with perforations or openings 26 and ribs 28. An upper flange 30 surrounds and extends outwardly from the respective side walls 22 a-d. A lid 32 is attached to body 20 by a hinge 34 which allows vertical movement of lid 32 into the interior of body 20 for retaining one or more tissue samples against bottom wall 24. Lid 32 is likewise formed with perforations 36 which may be elongated and generally separated by ribs 38. As shown best in FIG. 1, elongated perforations 26, 36 on the bottom wall 24 and lid 32 respectively extend toward central areas 24 a, 32 a of the bottom wall 24 and lid 32. This assists with allowing a mold (not shown) to fill with material, such as PFA, during the molding process of cassette 10.
Lid 32 is formed with a shape along its circumference which compliments the shape of side walls 22 a-d. In this regard, respective lengthwise side edges 40 a, 40 b of lid 32 are formed in the shape of a shallow “V” and each includes an apex 42 a, 42 b generally at the central portion thereof. This likewise corresponds to the lengthwise shallow “V” shape of side wall 22 a and opposite side wall 22 c. Thus, when the embedded tissue cassette is later placed in a microtome and sections are sliced from the embedded tissue cassette, the microtome blade will initially contact the apex corresponding of side wall 22 a or 22 c, depending on which side is facing up in the microtome. This has been found to increase the quality of ribboned slices being made from the embedded tissue cassette. That is, little or no fracturing of paraffin will occur at the paraffin/cassette interfaces.
Frame 12 more specifically includes an open interior 50 which receives cassette 10, and an angled front wall 52 which may be used for recording indicia, such as patient data. Respective upper and lower sets of detents 54, 56 and 58, 60 extend inwardly into the open interior 50 of frame 12. Initially, tissue cassette 10 is held between the upper pairs of detents 54, 56 by pressing the upper flange 30 downwardly past the upper sets of detents 54 and against the lower set of detents 56 after one or more tissue samples have been placed into cassette body 20 and lid 32 has been closed. Respective detents 61 extend inwardly from side walls 22 a-d to allow lid 32 to be snapped and retained in a closed position. During a staging operation, tissue cassette 10 will be moved vertically downwardly through frame 12 until flange 30 snaps past detents 58 and rests against detents 60 at a lower position (FIG. 5). Recesses 62, 64 are formed in at least two opposite side walls for allowing fingers of a gripper mechanism to register with the assembly during automated handling and embedding operations, as necessary. Preferably side walls 66 a, 66 b contain these recesses 62, 64, while additional side walls 66 c, 66 d may include additional structure allowing automated handling, or allowing other functions as necessary. Base mold 14 includes an open interior 70 for receiving frame 12 and may be surrounded by a resilient elastomeric seal 72 which prevents the escape of liquid paraffin during the embedding process. This eliminates an additional step of scraping off excess hardened paraffin from frame 12 after the embedding process is complete. Such excess paraffin can prevent the frame from properly fitting in a microtome chuck.
By comparing FIGS. 2 and 3 to FIGS. 4 and 5, it will be appreciated that during the staging process, tissue cassette 10 is moved vertically downward into the interior 70 of base mold 14 such that the bottom 24 of tissue cassette 10 contacts the bottom 74 of base mold 14. At this position, flange 30 of tissue cassette body 20 is received between respective detents 58, 60 holding it in this lower position. Frame 12 is preferably press fit and held frictionally within base mold 14 by one or more resilient seals 72. Seal 72 performs two functions in this embodiment. First, it physically holds frame 12 within base mold 14 by friction. This prevents the frame 12 and attached cassette 10 from floating or otherwise moving while base mold 14 is filled with paraffin. Second, it prevents the liquid paraffin from escaping from base mold 14 in the area between outer walls 12 a of frame 12 and interior 70 of base mold 14. It will be appreciated that a holding member other than seal 72 may be used instead to physically hold frame 12 against base mold 14. In this case, seal 72 may not be necessary. As some examples, such holding members may be clamps, fasteners, spring members or weights. In these figures, the tissue sample or samples within cassette 10 have been deleted for clarity. However, it should be understood, that lid 32 will be depressed down onto one or more tissue samples 80 contained with the interior of cassette body 20 (FIG. 6). While the assembly of cassette 10, frame 12 and base mold 14 is in the staged configuration shown in FIGS. 4 and 5, liquid paraffin is introduced through the open interior of frame 12 and through the perforations 26, 36 of cassette 10 into the interior 70 of base mold 14. The liquid paraffin is then preferably cooled on a suitable cooling device, such as a TEC, and the assembly of the tissue cassette 10, frame 12, embedded tissue sample 80 and hardened paraffin 82 is removed from base mold 14 as shown in FIG. 6. Frame 12 may then be used as a device to fix the assembly in a microtome chuck and slices are then taken from the bottom surface 82 a of paraffin 82. Initially, a facing blade is used to remove the initial layer of paraffin 82 and bottom wall 24 of tissue cassette 10. At this point, a different microtome blade may be used to take ribboned slices or shavings of tissue sample 80 and the surrounding paraffin 82 and side walls 22 a-d of tissue cassette body 20. Often times the same blade that is used for facing may also be used for sectioning.
FIGS. 7 and 8 illustrate one type of staging device 100 which may be used in conjunction with the tissue cassette 10, frame 12 and base mold 14 described above. Staging device 100 includes a handle 102 at an upper end and a staging mechanism 104 at a lower end. Staging mechanism 104 is coupled to a hollow cylinder 106 extending upwardly into a shroud 108 rigidly connected with handle 102. A plurality of four stabilizer members 110 are rigidly coupled with cylinder 106, while a plurality of preferably movable staging fingers 112 are coupled to a reciprocating shaft 114 which extends through cylinder 106. Shaft 114 is rigidly connected to handle 102 and is biased therewith in an upward position by a spring 116 positioned between a bottom surface 102 a of handle 102 and an upper surface 106 a of cylinder 106. Thus, it will be appreciated that stabilizer members 110 may be placed against an upper side of frame 12 for stabilizing purposes and handle 102 may then be depressed downwardly as indicated by arrow 118. This moves reciprocating shaft 114 and the attached staging fingers 112 downwardly against tissue cassette 10 thereby moving the tissue cassette 10 relative to frame 12 from its upper position shown in FIG. 3 to its lower, staged position shown in FIG. 5. To avoid pushing cassette 100 too far through frame 12, surface 108 a stops against surface 110 a as flange 30 reaches its lower position between detents 58, 60.
FIGS. 9 and 10 illustrate a second embodiment of a tissue cassette 120. Tissue cassette 120 includes a cassette body 122 having four side walls 124 a-d surrounding an open interior and bounded on a bottom side by a bottom wall 126. Side walls 124 a-d are constructed of ribs 128 separated by perforations 130 and bottom wall 126 is constructed by ribs 132 separated by perforations 134. Ribs 128 of side wall 124 a are offset in a lengthwise direction relative to ribs 128 of opposite side wall 124 b, as shown by distance “d” in FIG. 10. Distance “d” may vary, however, in this embodiment, on average, it is approximately 0.015″ to 0.030″. Offsetting ribs 128 in this manner ensures that a microtome blade passing through walls 124 a and 124 b contacts a more uniform amount of cassette material along its length. This leads to longer blade life, more uniform blade wear and more consistent high quality slices of embedded tissue. Ribs 132 and perforations 134 extend in their lengthwise direction toward a central area 126 a of bottom wall 126. In this preferred embodiment, side walls 124 a-d are constructed such that the ratio of plastic cassette material, such as PFA, to the open area formed by perforations 130 is approximately 3.7:1. To achieve at least this ratio in the preferred embodiment, ribs 128 have a width W1 of approximately 0.010″ to 0.014″ while perforations 130 have a width W2 of approximately 0.040″ to 0.050″. This perforated area, which is below solid side wall portion 136 is the area which will be cut with a microtome blade after embedding of tissue sample 80 (FIG. 6). Especially when using PFA with a Shore D hardness of 48 to 55 as the material for cassette 120, in combination with the industry standard paraffin embedding material mentioned above, this ratio of cassette material to open area has been found to improve the quality of cuts made through the embedded cassette and increase blade life significantly. Depending on the respective molecular weights of the embedding material and the cassette material, this ratio may be increased or reduced.
A flange 140 surrounds the upper side of cassette body 122 and contains respective upwardly facing depressions 142 a, 142 b, 142 c, 142 d. A number of downwardly facing depressions 142 e, 142 f (only two of several referenced) are also contained in flange 140. These depressions respectively register with at least detents 54, 56 and 58, 60 of frame 12 (see FIGS. 3 and 5). It will be appreciated that additional lower detents may be formed on frame 12 to provide further support and prevent cassette 10 from being pushed too far through frame 12. In such a case, additional depressions are formed in the underside of flange 140 to accommodate the additional supportive detents. The depressions allow the effective outward extension of cassette body 120 from frame 12 to be increased thereby increasing the number of slices that may be taken from the embedded cassette in a microtome. This can be important in many situations in order to obtain the desired slides for pathologic examination.
Detents 144 are also formed on side walls 124 a-d for retaining a lid 150 in place. Lid 150 is coupled to cassette body 122 by a hinge 152. Lid 150 is formed to be stiffer than bottom wall 126 of cassette body 122 so that lid 150 may be used to press the tissue sample against the bottom wall 126 and to press the bottom wall 126 against the rigid bottom 74 of base mold 14 (FIG. 5). Lid 150 is formed by a plurality of ribs 154 generally separated by perforations 156 and each extending in a lengthwise elongated fashion toward a central area 150 a of lid 150. Lid 150 further includes side edges 158 a, 158 b extending lengthwise thereon and having a shallow “V” shape with a central apex 160 a, 160 b. Angle α (FIG. 10) is preferably about 4°. As with the first embodiment, the shape of side edges 158 a, 158 b complements similar shape of side walls 124 a, 124 b of cassette body 122. In this regard, these side walls 124 a, 124 b, either one of which may be the initial wall cut in the microtome, include respective apecies 162 a, 162 b. Respective ends 164 a, 164 b of lid 150 also complement the similar shape of cassette body side walls 124 c, 124 d to achieve a close fit of lid 150 within cassette body 122. The use of cassette 120 in the process of embedding one or more tissue samples, including the staging process within frame 12 and the embedding or molding process within base mold 14 is the same as described above with regard to FIGS. 1-8.
FIGS. 11 and 12 illustrate a manual staging device 200 in accordance with a second embodiment. Staging device 200 comprises an upper handle 202 and a lower staging mechanism 204 coupled together rigidly by a shaft 206. In FIG. 12, handle 202 and shaft 206 have been deleted for clarity. Staging mechanism 204 preferably comprises a plurality of four staging members 210, 212, 214, 216 extending generally radially outward from shaft 206 and each including a finger portion 210 a, 212 a, 214 a, 216 a. The finger portions 210 a-216 a respectively engage upper corner portions of cassette 10 (FIG. 1) or cassette 120 (FIG. 9). When the user then pushes downward on handle 202, finger portions 210 a-216 a urge cassette 10 or cassette 120 downward into the lower, staged position (FIG. 5). To prevent the cassette from being moved too far downward, a pair of stops 218, 220 are provided on staging mechanism 204. In this embodiment, stop surfaces 218 a, 220 a stop against the upper surface of frame 12 as cassette 10 or cassette 120 reaches the lower, staged position (FIG. 5). Thus, the distance between the lower ends of finger portions 210 a-216 a and stop surfaces 218 a, 220 a equates to the distance between upper detent pair 54, 56 and lower detent pair 58, 60. It will be appreciated that other forms of staging mechanisms and stops may be provided while falling within the spirit and scope of the invention.
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U.S. Classification 422/400, 435/297.5
International Classification G01N1/36, B01L9/00, G01N1/31, B01L3/00
Cooperative Classification Y10T436/25, G01N2001/366, B01L2300/043, B01L9/52, B01L3/508, G01N2001/315, G01N1/36
European Classification B01L9/52, B01L3/508, G01N1/36
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:WILLIAMSON IV, WARREN P;BERKY, CRAIG B;WHITLATCH, STEPHEN P;AND OTHERS;REEL/FRAME:016024/0182