Chemistry: analytical and immunological testing – Including sample preparation – Stabilizing or preserving
Reexamination Certificate
2001-10-23
2004-03-02
Warden, Jill (Department: 1743)
Chemistry: analytical and immunological testing
Including sample preparation
Stabilizing or preserving
C436S174000, C436S043000, C436S046000, C436S047000, C436S164000, C436S169000, C436S178000, C435S325000, C422S050000, C422S051000, C422S068100, C422S051000, C422S063000, C422S064000, C422S065000, C422S066000, C422S082050
Reexamination Certificate
active
06699721
ABSTRACT:
This invention relates to laser capture microdissection (LCM). More specifically, this disclosure sets forth an improved film for use with laser capture microdissection in which the film is used in contact with the specimen and a coating is provided over the film which prevents non specific transfer of specimen without inhibiting desired laser capture microdissection.
BACKGROUND OF THE INVENTION
Laser capture microdissection (LCM) is known. Specifically, a sample such as a tissue specimen has multiple cell areas scattered throughout. Taking the case of a biopsy for cancer, these cell areas can include cancer cells, pre-cancer cells, irritated cells, normal cells and other tissue. The diagnostician usually desires only one type of cell or cell portion from one cell area—say for purposes of this example—the “pure” cancer cells. Further, the diagnostician requires sufficient quantity of the cells from the selected cell areas to perform further meaningful diagnosis. As a consequence, multiple samples of similar cells from one or more specimens are frequently required.
Laser capture microdissection is preferably performed with a transparent low temperature melting film such as (polyethylene/vinyl acetate), CAS 24937-78-8) (also known as EVA) manufactured by the Du Pont Corporation of Wilmington, Del. The reader will understand that many other manufacturers vend similar materials.
Specifically, a specimen—usually on a glass slide is overlaid with the low temperature melting film such as EVA. Thereafter, a cell area of the sample wanted for dissection is visualized and located, preferably through a microscope. When location has occurred, precision heating and melting of the low temperature film occurs overlying the visualized cell area. The precision heating of the low melting point film occurs by having a laser heat that portion of the low melting point film that overlies the cell area targeted for microdissection. At the precision heated area, flow of the melted EVA occurs from the film to the sample onto the visualized cell area. The sample is adhered to the melted EVA upon solidification of the precision melted portion of the film. Thereafter, the film is removed from the sample, preferably by placing the film overlying the specimen under tension and lifting the film away from the sample. Typically, the sample at the adhered identified cell area sticks to the film with the result that a microdissection occurs. The reader will understand that the film a coating can either be stretched or can reside on a support surface.
In LCM, at least a major part of the mechanism of adhesion is mechanical. The heated film overlying the selected cell area flows into and around the specimen portions to be microdissected. Thereafter, the heated and flowed low temperature melt film solidifies. When the film is withdrawn, the physical interference between the flowed and solidified film material and the cell area of the specimen intended for microdissection causes the film when it is withdrawn to “pull” the selected cell area from the remainder of the specimen. Microdissection occurs.
The primary reason for low temperature in the melting of the film is to avoid damage to or change the nature of the specimen. EVA, among plastics has a uniquely low melting range, which can be controlled by the manufacture. Such manufacture control can include the addition of a variety of ingredients (e.g. rubber) to adjust the melting point and other properties. The ethylene part of the polymer can be used for property variations.
Understanding this mechanical adhesion, the reader can quickly understand the reason for using a low temperature melt film. It is obviously desired to remove the targeted portion of the specimen for further diagnosis. Where the melted film flows in and around the targeted portion of the specimen, undue heating changes the nature of the targeted cellular material and makes may invalidate subsequent analysis or diagnosis. This subsequent analysis, or diagnosis, includes potentially a variety of methods for research and clinical evaluation, such as genetic, immunological, enzymatic, and protein analysis.
At the same time, precise and precision transfer of the intended cell area is required. Such precise transfer must gather only the identified cell area—say for the sake of the example discussed above—the “pure” cancer cells. In the LCM, recovery of materials from cell areas other than cellular material within the identified cell area is referred to as “non-specific transfer.” Non specific transfer can be detrimental to further analysis including biological amplification techniques such as PCR. Some applications of LCM are sensitive to very low levels of undesired sample areas.
To avoid non-specific transfer, LCM as currently practiced has divided itself into two broad techniques. In one technique, known as non-contact LCM, the film to which attachment occurs is held spaced a small but constant interval from the sample. When local heating of the EVA or other film utilized occurs, the film expands across the spatial interval, and adheres to the specimen at the visualized portion. When the film is removed, microdissection occurs. A solution to this problem has been to devise various means of spacing the EVA film away from the tissue, so that the EVA contacts the tissue only in the selected spots by its expansion during laser melting.
The EVA expands in a column or pedestal at the area of activation by about 10% to 15%, melts into the tissue sample, and then withdraws away slightly, retaining a microscopic tissue sample upon cooling. The desired spacing of the EVA surface away from the tissue sample is of the order of 10 micrometers. There are various ways of providing the spacing, including putting a spacer film on the EVA, located so the spacer film is in contact with an area of the tissue away from the desired sampling point and holding the EVA surface away from the tissue. With regard to such spacing techniques, non specific transfer may also occur even though a spatial interval is present, due to loose or weakly adhering substances and unevenness of tissue.
It is desirable to press the surface of the EVA against the tissue sample with controlled force, as one of the control parameters of tissue sampling, with the spacing preventing actual contact of the EVA and tissue. The coating of this disclosure may be desirable to prevent non specific transfer in this method also.
It is to be understood that non-contact microdissection is not without problems. Specifically, maintaining the film at a precise closely spaced interval from the specimen at the visualized cell area is difficult. Precision control of the parameters of contact of the low temperature melt film to the sample is difficult.
In another technique of LCM, which is directly applicable to this invention, the film is brought into direct contact with the specimen before melting occurs. In the past, this direct contact with the specimen has caused non-specific transfer. Specifically, the film used—usually EVA—is naturally tacky. This natural tackiness results from the softening point of the film that is required to minimize damage to the microdissected cell area being removed.
Complicating LCM, the biological specimen is also non-homogenous. The specimen typically contains proteins, carbohydrates, fats oils and other cellular materials in an irregular matrix. Portions of this irregular matrix can preferentially adhere to tacky surfaces of the film without the laser heating. Thus, when the targeted material is adhered in the LCM process and withdrawn from the specimen, undesired adjacent cell areas of the specimen are removed and transported by the film. It is this non-specific transfer which it is the purpose of this invention to avoid.
SUMMARY OF THE INVENTION
A low temperature melt film such as EVA is prepared for laser capture microdissection by having a thin specimen non-adhering coating which is hard and non tacky. The hard and non-tacky coating may be in the range of 0.1% to 10% of the total film thickness placed on the sample exposed
Gorrindo Tristan
Peterson John I.
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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