Patent Publication Number: US-8530808-B2

Title: Microwave-assisted heating and processing techniques

Description:
FIELD OF THE DISCLOSURE 
     Aspects of the disclosure are related to processes and apparatus for processing tissue, e.g., histological processing. 
     DESCRIPTION OF BACKGROUND INFORMATION 
     Histological tissue processing is an important part of many different medical and forensic situations. The processing is performed in order to create stained cellular structures on a microscope slide for subsequent study and analysis using a microscope. By way of example, a doctor may perform a biopsy, and remove a small sample of tissue from a patient. This tissue is then placed into a small container of preservative, i.e., a fixative. At the lab, a histotech removes the specimen from the fixative, and places it into a labeled cassette. 
     The specimen is now processed, either traditionally (i.e., without acceleration techniques such as the use of a microwave) or via microwave processing, to produce a stained version of the tissue. 
     In one example of microwave tissue processing, cassettes containing formalin-fixed tissue may be place into a rack, and the rack is then placed into a Pyrex™ dish of ethyl alcohol. This is the dehydration step. In a next step, “clearing”, isopropyl alcohol is applied to the tissue, which removes the remaining water, the ethyl alcohol, and fats from the tissue. In a next step, an embedding agent (e.g., Paraffin) is infiltrated and embedded into the tissue. Different agents may be used depending upon the protocol. For example, rather than use isopropyl alcohol, isoparaffinics may be used. In addition, the fixation of the tissue may be carried out by means other than with the use of formalin. For example, microwave-assisted fixation may be used with different agents such as a glyoxal fixative. 
     Other processing methods include, e.g., tissue processing for electron microscopy and heating or drying of slides containing specimens, to name just a few. 
     SUMMARY OF THE DISCLOSURE 
     In accordance with one aspect of the disclosure, apparatus are provided, which include a specimen vessel holding at least one biological tissue sample. A histological agent is in the specimen vessel so as to come into contact with the tissue sample. A microwave susceptor structure is provided in, near, or integral to the specimen vessel. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       Embodiments of the disclosure are further described in the detailed description which follows, by reference to the noted drawings, in which like reference numerals represent similar parts throughout the several views of the drawings, and wherein: 
         FIG. 1  is a flow diagram of various steps in a tissue processing system; 
         FIG. 2  illustrates a side schematic view of a specimen batch container; 
         FIG. 3  is a block diagram of a tissue processing system; and 
         FIG. 4  is a sectional side view of another example of a processing container. 
     
    
    
     DETAILED DESCRIPTION 
     Referring now to the drawings in greater detail,  FIG. 1  is a flow diagram of an example process carried out in a histological tissue processing environment. In act  20 , the specimen is accessioned. At this point, specimens are given a number which will identify each specimen for each patient. In a next act  22 , the tissue that has been removed from the body for diagnosis is examined, for example, by a pathologist, a pathology assistant, or a pathology resident. Gross examination may include describing the specimen and placing all or parts of the specimen into a plastic perforated cassette which holds the tissue while it is being processed. 
     In a next act  24 , the tissue is fixed. Fixation preserves the tissue in as life-like state as possible, and should be carried out as soon as possible after the tissue is removed to prevent autolysis. Formalin (formaldehyde) is the most commonly used fixative. Other groups of fixatives include aldehydes, mercurials, alcohols, oxidizing agents, and picrates. 
     In act  26 , the tissue is dehydrated. This may be done, for example, with a series of alcohols. 
     In a next act  28 , the dehydrant substances are removed from the tissue. This may, for example, be done with xylene, or in the case of microwave processing, with other safer substances, such as isopropyl alcohol. After the clearing act  28 , the tissue is infiltrated and embedded with an embedding agent, typically paraffin. Different types of paraffin can be used, for example, that have different melting points and different degrees of hardness. One product called paraplast contains plasticizers that make the paraffin blocks easier to cut. The infiltration and embedding process can be assisted by applying a vacuum to the container holding the tissue. The process, including dehydration at act  26 , clearing at act  28 , and embedding at act  30  (and also including fixation at act  24 ) can be automated. 
     Subsequent to infiltration and embedding in act  30 , the tissue is sectioned at act  32 , at which point the tissue is cut into sections that can be placed on a slide. This may be done with a microtome. After the sectioning, de-embedding is performed at act  34 . This prepares the tissue for subsequent staining at act  36 . After the tissue is stained, cover slipping is performed at act  38 , at which point the stained section is covered with a thin piece of plastic or glass to protect the tissue from being scratched, and to provide better optical quality for viewing under the microscope. 
     As shown in  FIG. 1 , in the processes and systems of the present disclosure, one or more of the fixation act  24 , the dehydration act  26 , the clearing act  28 , the infiltration and embedding act  32 , the de-embedding act  34 , and the staining act  36 , may involve applying a microwave to the tissue during those acts. Per variations of the embodiments of this disclosure, the microwave may be used to accelerate the reaction, to control the temperature of the tissue and of reagent, to increase the temperature of the reagent (for example, in the case of infiltration and/or embedding an embedding agent into the tissue at act  30 ), and/or to enhance, inhibit, or otherwise affect certain biological functions of the biological tissue sample for subsequent study. 
     When applying a microwave to the tissue sample, if only the tissue sample and the vessel holding the tissue sample (e.g., a slide, a container, a conduit, etc.) are polar substances (i.e., they include no other polar materials, with a high dielectric constant—e.g., only paraffin), the microwave&#39;s reaction chamber may not be sufficiently attenuated. This can cause problems in the operation of the microwave oven, or it could cause damage to the biological tissue itself, which may overheat and be damaged. As shown in  FIG. 1 , any one or more of these acts (particularly, e.g., infiltration, embedding, de-embedding, cleaning using isoparaffinics, and slide drying) may involve the use of a microwave  40 , while the polar substances  44  in the microwave reaction chamber may be limited. Accordingly, to reduce the risk that the tissue will be damaged, a microwave susceptor material  42  may be provided in the reaction chamber to cause attenuation of the microwaves and to prevent damage to the tissue during such acts. 
     This material  42  is helpful, e.g., during the infiltration and embedding act  30 , as well as the de-embedding act  34 , at which point the only materials in the specimen batch container (other than microwave susceptor material  42 ), and accordingly, in the reaction chamber of the microwave, may be the tissue sample itself and paraffin (which is a non-polar substance). Should microwaves be used to heat the paraffin without the presence of susceptor material  42 , to thereby liquefy the paraffin for either infiltration and embedding or for deparaffinization, the biological tissue itself could be damaged. Accordingly, microwave susceptor material  42  is provided in the microwave chamber, specifically, in certain embodiments, in the specimen batch container, to heat up the paraffin to a desired temperature while the specimen is not damaged. 
       FIG. 2  shows one example embodiment of a specimen batch container  50 . The view shown in  FIG. 2  is a schematic side view. The container may, for example, be a tray or a nine inch by nine inch Pyrex™ dish. The illustrated specimen batch container  50  includes a microwave susceptor sheet  54 , which, in this illustrated embodiment, spans substantially the entire surface of the bottom of container  50 . A set of specimen containers  52  is provided. In the embodiment, each container corresponds to a unitary tissue sample, and includes perforations. The containers may be, for example, cassettes with hinged covers, arranged and held by a cassette rack. 
     The illustrated microwave susceptor sheet  54  may comprise a microwave susceptor material or structure, and need not be a sheet. The microwave susceptor material may be configured to apply directional heat when in a microwave reaction chamber. The illustrated microwave susceptor material includes metalized film. It may be coated with a protective polyester layer. The illustrated metalized film includes aluminum, which may be in the form of aluminum flakes. Alternatively, the microwave susceptor material could comprise, for example, the material as disclosed in U.S. Pat. No. 5,021,293, or as described in U.S. Pat. Nos. 4,518,651, 4,267,420, 4,434,197, 4,190,757, 4,706,108, U.K. Patent Application No. 2,046,060A, or European Patent Application Publication No. 63,108, the content of each of which is hereby incorporated by reference herein (except that any terms as coined or defined in such references shall not affect the meaning of such terms herein or in the appended claims hereof). While each of these other materials could be, for example, used as alternatives, the use of a material having a metalized aluminum flake layer is one embodiment. Example aluminum materials are described in the detailed description of the noted U.S. Pat. No. 5,021,293. 
     The illustrated specimen batch container  50  shown in  FIG. 2  further includes a histological agent  53 . The histological agent  53 , depending upon the stage within the tissue process being performed, may include one of a dehydrating agent, a clearing agent, an embedding agent, a de-embedding agent, and a staining agent. In the case of the use of microwaves to assist in the embedding or the de-embedding, i.e., as per acts  30  or  34  as shown in  FIG. 1 , the histological agent may include paraffin, or a type of paraffin. The paraffin is placed in the specimen batch container so as to come into contact with the tissue samples when the histological embedding agent is treated. The way it is treated is generally by heating the paraffin. In certain contexts, paraffin may be heated, for example, to 82 degrees celsius. The paraffin is liquified to facilitate infiltration and embedding of the paraffin into the tissue sample or to facilitate the de-embedding of the paraffin out of the tissue sample. A vacuum may be applied to the processing station in order to facilitate infiltration and/or embedding of the paraffin into the tissue sample. 
       FIG. 3  is a block diagram of an example tissue processing system in accordance with one embodiment of the present disclosure. The illustrated tissue processing system  59  may be a manual tissue processing system, which provides for manually carrying out one or more aspects of the tissue processing process. Alternatively, the system may be partially automated, or fully automated. The illustrated system  59  includes a station  60 , at which a batch container  62  is placed. In the illustrated embodiment, there is only a single station  60 , and that station is stationary and is located within the reactive chamber of a microwave unit  61 . Batch container  62  includes a set of specimen containers  64 , and is supported by a batch container support structure  66 . 
     Optionally, a positioning mechanism  68  may be provided, to physically place a particular batch in a batch container  62  in station  60 , to remove a given batch container  62  from station  60 , and to replace or otherwise position batch containers in or out of microwave unit  61 . Given that the specific example embodiment includes only a single station  60 , a positioning mechanism  68  is generally not necessary. 
     A microwave susceptor structure  70  is provided in or as part of the structure of batch container  62 . The illustrated microwave susceptor structure  70  may include a metalized layer or film. It may further include such a film laminated to a paper-based card stock. In addition, the microwave susceptor structure  70  may include such a film coated with a protective polyester layer. The metalized film may include aluminum. The microwave susceptor structure  70  is in the reaction chamber of microwave unit  61 . In the illustrated embodiment, it is in the bottom of batch container  62 . In the embodiments illustrated herein, microwave susceptor structure  70  is in direct or indirect contact with the histological agent that is within the batch container  62 . The microwave susceptor structure  70  may be fixed to, embedded within, or integral to the structure of specimen batch container  62 , or, it may be fixed to, embedded within, or integral to the structure of the specimen containers  64 . Alternatively, it could be a disposable separate sheet of microwave susceptor material, e.g., placed in the bottom of batch container  62 . 
     One or more sensors (including temperature sensors)  71  may be provided for sensing various conditions within batch container  62 . For example, the temperature of substances within batch container  62  may need to be sensed in order to facilitate control of various aspects of the tissue processing techniques, which will be carried out by a programmable control mechanism  72 . In the illustrated embodiment, programmable control mechanism  72  includes a computer, for example, a personal computer, which is configured (e.g., provided with software programmed) to automatically process tissue, including carrying out fixation, dehydration, clearing, and infiltration and embedding, all in the processing station  60  in the reaction chamber of microwave unit  61  without the need to move or remove the batch container  62  during or in between any of the acts of fixation, dehydration, clearing, and infiltration and embedding. 
     The illustrated system  59  further includes a substance transfer mechanism or a plurality of such mechanisms  74  for facilitating the transfer of substances (mainly the histological agents) into and out of batch container  62 . Other treatment mechanism or mechanisms  76  may be provided for treating or acting on batch container  62 . For example, a mechanism may be provided for applying an agitation to batch container  62  and/or applying a vacuum to the container. Each of substance transfer mechanism(s)  74 , other treatment mechanism(s)  76 , optionally provided positioning mechanism  68 , sensor(s)  71 , and microwave unit  61 , may be connected to programmable control mechanism  72  via an interface to allow the programmable control mechanism  72  to be aware of the status and functioning of each of those subsystems as well as to send control signals and control the operation thereof. 
     The sensor(s)  71 , programmable control mechanism  72 , microwave susceptor structure  70 , and microwave unit  61 , together, can be configured to serve as a heat sink or temperature buffer, i.e., to maintain physiological temperatures as desired while allowing microwave stimulation of a specimen, e.g., of a living tissue sample in one embodiment. In addition, a microwave unit and susceptor material may be used to cause slide drying, and/or de-embedding (as noted above). 
       FIG. 4  is a sectional side view of a processing container  80  (a batch container as described in the previous embodiments). The illustrated processing container  80  may include a cover  81  as shown, and holds a plurality of specimen containers  83 . In the bottom of container  80 , a microwave susceptor structure  82  is embedded. The embedded microwave susceptor structure  82  may be permanently embedded into processing container  80 , or it may be removable into and out of a recess, for example, shown as a recess  86  shown at the right hand end of the illustrated processing container  80 , in the view shown in  FIG. 4 . Microwave susceptor structure  82  is embedded within the recess  84  provided in the bottom portion of the illustrated processing container  80 . 
     The illustrated microwave susceptor structure  82 , and/or the susceptor material  54  as shown in  FIG. 2 , and/or the microwave susceptor structure  70  as shown in  FIG. 3 , can be “tuned” by having particular patterns. For example, the structure of the susceptor could include slats, like that of a venetian blind, or overlapping radiating “spoke” patterns. The slats or spokes could be moved in order to dynamically change or tune the microwave susceptor structure. 
     Histological processing for electron microscopy involves steps similar to those as described above for the histological context, generally using different histological agents. For example, an embedding agent may include an epoxy resin instead of paraffin. 
     While certain embodiments described above include a specimen batch container containing a set of perforated specimen containers, one or more biological tissue samples may reside in, on, or otherwise be held by any specimen vessel appropriate for the circumstances. A few example specimen vessels include a container, a plate, an open fluid holder, a closed fluid holder, a slide, a conduit, a combination of any such structures, etc. 
     In one or more embodiments, the microwave unit may include a lab microwave with enhanced temperature control features. For example, the unit may have shorter pulse cycle times (e.g., on the order of a second, several seconds, or less per cycle), a power amplitude adjustment mechanism, and/or change controls to change, e.g., the cycle time, pulse time within a cycle, the power amplitude, and/or one or more other parameters in order to control the energy and/or heating effects of the unit on items within the chamber. 
     The claims as originally presented, and as they may be amended, encompass variations, alternatives, modifications, improvements, equivalents, and substantial equivalents of the embodiments and teachings disclosed herein, including those that are presently unforeseen or unappreciated, and that, for example, may arise from applicants/patentees and others.