Chemistry: analytical and immunological testing – Including sample preparation
Reexamination Certificate
2000-10-30
2004-04-13
Warden, Jill (Department: 1743)
Chemistry: analytical and immunological testing
Including sample preparation
C436S043000, C436S063000, C435S325000, C422S050000, C422S063000, C422S064000, C422S065000, C422S066000, C422S068100
Reexamination Certificate
active
06720191
ABSTRACT:
This invention relates to laser capture microdissection (LCM) in which direct extraction of cellular material from a tissue sample occurs to a transfer surface. The disclosure herein relates to isolating selected tissue samples to a film matrix in a format where the isolated sample segments can be conveniently collected for subsequent analysis.
BACKGROUND OF THE INVENTION
Laser Capture Microdissection (LCM) has been described in Science magazine (Nov. 8, 1996 and Nov. 21, 1997). Summarizing, LCM typically involves placing a transporting substrate having an activatable coating for adhering to identified cellular material on a tissue sample. For example, a large (several cm
2
) piece of transparent, thermally activatable adhesive film (e.g. EVA polymer) is placed in close apposition with the upper tissue surface of a standard (desiccated) histopathology section (5-15 um thick) mounted on a glass microscope slide.
Once the identified cellular material of the tissue sample for micro-dissection is selected, typically by examination under a microscope, the substrate is then activated, typically be being pulsed with laser beam. The light energy is absorbed by the plastic film which melts in a small region. The activatable coating then flows onto and around microscopic tissue components thereby causing the film on cooling to be firmly adherent to the identified cellular material of tissue sample. Other target areas on the same slide can similarly treated.
When the film is subsequently lifted off the slide, the selected tissue comes with the film in a series of spots leaving the other tissue behind on the slide. Unfortunately, just as the identified cellular material was dispersely located on the slide, the identified cellular material is dispersely distributed on the transporting substrate.
In the ordinary case, the selected series of spots is then removed from the film by placing the film in a suitable reagent (e.g., proteinase K which digests the structural proteins of the tissue) which frees the molecules of DNA or RNA to be subsequently analyzed (using, e.g., PCR and gel electrophoresis). In order to confer specific transfer of single targets to a given reagent solution (i.e., specific “molecular extraction buffers”), the transferred tissue spots and underlying attached film can be manually excised with a scalpel or scissors, or punched out with a precision punch/die directly into a cuvette.
With the development of this technique, demand has arisen for refinement. Accordingly, and in the text that follows, we identify the requirements of LCM and thereafter propose solution to those requirements. It is to be understood that in setting forth the requirements for laser capture microdissection, we claim invention. It goes without saying that the recognition of the problem to be solved can constitute part of the invention along with the solution to the problem once it is recognized.
In LCM, there is potentially a great research need (and eventual clinical need) to transfer smaller and smaller spot sizes (e.g. 25 microns or less). This leads to transfer of selected single cells or even organelles in order to study molecular modifications of specific cells within a pathology or cytology specimen. This poses two technology problems:
1) the accurate targeting, and adhesion of the film to specific cells based on microscopic observation, and
2) keeping the specificity of transfer high by minimizing the adjacent area of film transferred into the molecular extraction buffers.
In LCM, the spots in the film are randomly located roughly in a position corresponding to their location on the sample. When smaller spot sizes of sample are extracted, a problem which arises is finding them on the removed piece of film or transfer surface. Precise location is required so that the extracted portions of the sample can be precisely cut out. With precision cutting, the target tissue is completely recovered with minimal contamination from surrounding areas of film with low density nonspecific tissue adhesion.
Restricting LCM to the disclosed technology, precise computer control of the storage position and storage of coordinates may be practical. However, this computer control may loose the reliability of visual observation of the transfer process and may be complex and expensive. Further, computer location could fail to be accurate in many cases such as when the tape is distorted when it is lifted off the slide.
Nonspecific transfer to the film of un-targeted tissue in the region surrounding the identified cellular material is another problem. This non-specific transfer becomes increasingly important as spot size of targeted tissue is reduced. Ideally, no tissue outside the targeted region illuminated by the laser should be attached to the film upon its removal from the slide. Attachment of any undesired tissue would cause sample contamination with the desired tissue. As smaller and smaller target spots are used, the amount of stray tissue which can be tolerated becomes proportionally smaller.
Presuming that an extremely small contact area with a sample can be achieved, a problem then arises as to how to handle the transfer surface. Specifically, the positioning of a small spot of activatable transfer surface on the tissue at the target site, picking the small activated transfer surface off the tissue, and placing the isolated targeted cells in a cuvette, or storing them in a specifically identifiable manner without contamination by un-targeted tissue elements.
Having set forth the requirements, we now turn attention to a solution.
SUMMARY OF THE INVENTION
A method and apparatus of gathering by LCM identified cellular material from random locations on a tissue sample to designated locations on a transporting substrate enables convenient further processing. A transporting substrate has an identified mapped location for receiving identified cellular material. At least a segment of a selectively activatable coating is placed on the side of the transporting substrate in apposition to the tissue sample at the mapped location. The transporting substrate and sample are relatively moved to place the selectively activated coating at the mapped location in apposition to identified cellular material of the tissue sample which is to be extracted. Thereafter, the selectively activating coating is activated and impressed or impressed and activated to form an adhesive region on the transporting substrate for adhering to the identified cellular material. Upon removal of the transporting substrate from the tissue sample, identified cellular material adheres to the transporting substrate at the mapped location.
In a first embodiment, individual small pieces or coatings of selectively activated material are placed on a transporting film. For example, a selectively activatable coating is placed at the center of discrete pieces of film and exposed toward the sample. Each different piece of film is separately activated at its coated center by the laser. Apposition to the tissue during or after laser action occurs by using a pressure plate.
In an alternate embodiment, a continuous strip including a transparent substrate is used to hold the film, such as a continuous reel of tape with equally spaced, centrally located pieces of selectively activatable coatings. Thereafter, the continuous strip of transparent substrate is incrementally advanced so that center of the activatable coating is in the center of the microscope field. Adjacent bar codes or other optical encoded identifiers could serve to identify the individual transferred samples.
Sample collection can include a pressure plate actuated to hold the transporting substrate in contact with the tissue in the center of the microscope field before or after laser heating. After laser heating and attachment to the selected material from the sample, the pressure plate is raised, and the transporting substrate with the local activated coating with adherent tissue separated from the tissue sample. This process is sequentially repeated so that the transporting substrate is again advanc
Bonner Robert F.
Goldstein Seth R.
Peterson John
Pohida Thomas
Smith Paul D.
Hynes William Michael
Sines Brian
The United States of America as represented by the Department of
Townsend and Townsend / and Crew LLP
Warden Jill
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