Isolation of cellular material under microscopic visualization

Technical Field The present invention relates to methods and devices for molecular analysis of cell samples. More specifically, the present invention has been used in combination with many different techniques that allow the analysis of proteins, such as enzymes, as well as mRNA and DNA from substantially pure populations and subpopulations of a particular cell type. The method and apparatus for microremoval and molecular analysis of the resulting cell sample. The present invention further relates to libraries made from cellular materials that are directly extracted in the methods of the invention. Background Techniques Many diseases are currently understood at the molecular and genetic levels. Analysis of such molecules is important for disease analysis and prognosis. Previous methods for the direct extraction of tissue material from tissue samples are limited because the extraction represents only the average content of disease-related markers. In practice, the tissue is very heterogeneous, and most diagnostic parts of the tissue can be restricted to hundreds or less of cells in the lesion. Normal tissue samples contain various cell types that surround and flank preinvasive and invasive tumor cells. 1. Areas of tumor tissue subject to undergo biopsy and diagnosis as small as 0 mm can include normal epithelium, pre-invasive stage of cancer, field cancer, invasive cancer and inflammatory areas. As a result, conventional exfoliation and cleavage methods will collect all cells of those types, and therefore the loss of alleles will be masked by the presence of normal copies of alleles in contaminating non-malignant cells. Analysis of genetic results by those conventional methods is always plagued by contaminating alleles from normal, unwanted or vascular cells. Molecular studies of human tumors are currently limited by the techniques and model systems available for their characterization. Studies for quantitatively or qualitatively assessing protein or nucleic acid expression in human tumor cells are compromised by the various cell populations present in large numbers of tumor specimens. The histological field of invasive tumors typically represents many cell types, such as tumor cells, substrate cells, endothelial cells, normal epithelial cells and inflammatory cells. Tumor cells often make up a relatively low percentage of the total cell population, making it difficult to interpret the significance of net protein or nucleic acid changes in those specimens. The process of tumor invasion and metastasis depends on the enhanced proteolytic activity of the invading tumor cells. Matrix metalloproteinases, cathepsins B, D and L, and plasminogen activators are involved in the metastatic cascade. Cathepsin D has been shown to be an independent marker of progression in breast cancer. Several strains of interrelationships support the notion that proteases are important in tumor invasion: Increased protease activity in highly metastatic tumor cell lines and / or altered non-cell distribution of proteases, enhanced protease expression in invasive human tumors as determined by immunohistochemistry and assay of tumor tissue homogenates , And elevated protease mRNA levels in human tumors. All of these techniques produce important information about protease expression in human tumors, however, they do not provide a definitive phenomenon in which proteases are up-regulated in specific areas where tumor invasion occurs. .. Studies of human tumor cells in culture do not explain the complex interactions between host cells and extracellular matrix and tumor cells, and how they can regulate tumor cell protease productivity or activation. Immunohistochemical staining allows testing of enzyme distribution in the area of tumor invasion, however, results vary with tissue fixation and antibody-antigen affinity, and provide only semi-quantitative assessment of protein levels. To do. In addition, the quantitative interpretation of staining results is complicated by the variability of staining patterns within tissue fragments, the subjective assessment of staining intensity, and the difficulty in interpreting the significance of substrate staining. In addition, many antibodies used in protease studies do not differentiate proenzymes from reactive enzyme species. Assays at the enzyme or mRNA level from homogens of human tumors do not explain any of the concomitant pathophysiological processes that can occur in the mixed cell population or tissue within the specimen. Human tumors accumulate genetic abnormalities as they progress from a single transformed cell to invasive and metastatic cancer. Identification and characterization of genes that are mutagenic, deleted or aberrantly regulated can provide important insights for cancer diagnosis, prognosis, and treatment. In addition, identification of such genetic damage can facilitate early diagnosis by definitive identification of premalignant damage so that they can be treated before they progress to invasive cancer. The general view of cancer progression is that cells are in the tumor suppressor gene It means that it can be transformed after obtaining two separate changes. Subsequent tumors gradually progress from dysplastic injuries to invasive and metastatic tumors. Field cancers have sometimes been observed to occur in association with widespread epithelial hyperplasia, and larger invasive tumors are often associated with areas of cancer around the tumor. Pathologists histologically interpret the close association of atypical hyperplasia, field cancers and invasive tumors as a phenomenon of cause-effect relationships between real things. However, there have been few direct phenomena supporting this model so far. Conventional research methods do not allow researchers to test genetic alterations in preinvasive injury. Even the most sophisticated genetic testing techniques to date are of limited value, as the input DNA, RNA or protein to be analyzed does not result from a pure cell population exhibiting disease morphology. Several methods for tissue microexcision have been reported to address this issue: Overall incision of frozen tissue block to enrich specific cell populations, irradiation of manually ink-stained fragments to destroy unwanted genetic material, palpation preparation of frozen tissue specimens, and microexcision with manual instruments .. However, these methods are not sufficiently accurate and effective for conventional research or high volume clinical molecular diagnostic applications. For example, manual microexcision has good accuracy, but is time consuming, labor intensive, requires a high degree of manual dexterity, and is generally not suitable for ordinary technicians. Absent. The present invention provides novel and improved means for specifically testing genetic alterations in preinvasive lesions of common epithelial tumors, such as breast and prostate cancers. In particular, the present invention allows microsampling of as few cells as one, along with RNA and DNA extraction of sampled cells. This method has been shown to be very sensitive and more than double the size of previous and current techniques. It is a sensitive detection of heterozygous deletions in early preinvasive injury, which is the way to discover new loci on chromosome 11 for breast cancer and new loci on chromosome 8 for prostate cancer. Made possible. Implementations of the present invention further allow the production of gene libraries from extracted materials. Thus, libraries from cells of interest, especially abnormal cells, are made and can be compared to libraries made from other cells in the immediate vicinity or adjacent, such as normal cells. Such libraries, for example, compare one or more specific loci, compare the expression of one or more RNAs, especially mRNA, isolate and / or clone one or more specific nucleic acids. Can be used to and for similar things. Disclosure of the Invention It is therefore an object of the present invention to provide a method for identifying specific cells in a cell tissue sample. Another object of the present invention is to provide a method for directly extracting specific cells from a cell tissue sample. Fine It is a further object of the present invention to provide an automated method for identifying specific cells in a cell tissue sample. It is a further object of the present invention to provide an automated method for the direct extraction of specific cells from a tissue sample. A further additional object of the present invention is to provide a method of obtaining a pure cell population from a tissue sample. According to those and additional objectives of the invention that will become apparent as the description of the invention progresses, the invention provides a method of direct extraction of cellular material from tissue samples, where this method is used. , A) Slide-Supply a fixed tissue sample; b) Form a cell image field of the tissue sample using a microscope; c) Identify at least one compartment of the cell of interest from the cell image field And that cells of at least one compartment of interest contain a different type of cell than adjacent compartments of cells; and d) extract cells of at least one compartment of interest from the tissue sample; Include. In another aspect, the invention provides a method of directly extracting cell material from a tissue sample, where the method a) feeds the tissue sample; b) provides an adhesive selection region. Contacting the tissue sample with a selectively activable surface that can be activated to provide; c) identify at least one portion of the tissue sample to be extracted; d) said at least 1 of the tissue sample By selectively activating a region of the transfer surface that corresponds to and is in contact with that portion, the activated region of the transfer surface becomes the at least one portion of the tissue sample. Selectively adhere; and e) said at least one portion of the tissue sample while maintaining adhesion between said activated region of the transfer surface and said at least one portion of said tissue sample. Includes separating the transfer surface from the tissue sample so that it is extracted from the rest of the tissue sample; Including that. In another aspect, the invention provides a method of directly extracting cell material from a tissue sample, where the method a) feeds the tissue sample; b) provides an adhesive selection region. Contacting the tissue sample with a selectively activable surface that can be activated to provide; c) identify at least one portion of the tissue sample to be extracted; d) said at least 1 of the tissue sample By selectively activating a region of the transfer surface that corresponds to and is in contact with that portion, the activated region of the transfer surface becomes the at least one portion of the tissue sample. Selectively adhere; and e) said at least one portion of the tissue sample while maintaining adhesion between said activated region of the transfer surface and said at least one portion of said tissue sample. Includes separating the transfer surface from the tissue sample so that it is extracted from the rest of the tissue sample; Including that. In another aspect, the invention provides a method of directly extracting cell material from a tissue sample, where the method a) feeds the tissue sample; b) provides an adhesive selection region. Contacting the tissue sample with a selectively activable surface that can be activated to provide; c) identify at least one portion of the tissue sample to be extracted; d) said at least 1 of the tissue sample By selectively activating a region of the transfer surface that corresponds to and is in contact with that portion, the activated region of the transfer surface becomes the at least one portion of the tissue sample. Selectively adhere; and e) said at least one portion of the tissue sample while maintaining adhesion between said activated region of the transfer surface and said at least one portion of said tissue sample. Includes separating the transfer surface from the tissue sample so that it is extracted from the rest of the tissue sample;

BRIEF DESCRIPTION OF THE DRAWINGS The present invention will be described with reference to the accompanying drawings provided by non-limiting examples only. Figure 1 shows how tissue samples are microscopically visualized and displayed on a display monitor, and how the area of the visualized sample is followed for microexcision and analysis. It is a schematic of a functional system showing a microscope. FIG. 2a-2c is a set of functional system schematics showing how tissue sample compartments are extracted from slide-fixed tissue samples according to one aspect of the invention. Figure 3 illustrates another device for extracting sample compartments from slide-fixed tissue samples. 4a and 4b are illustrations of a manual extractor manipulator that can be used with the extractor of FIG. 3 of the present invention. FIG. 5 is a functional system schematic showing how the sample tissue compartments can be directed to the appropriate analytical protocol. Figures 6a and 6b show the expression of MMP-2 in 10 invasive colon cancer cases (Figure 6a) and 5 invasive breast cancer cases (Figure 6b) compared to normal colonic mucosa from the same patient. Is shown. Figure 7 shows SSCP analysis of the MMP-2 activation site. FIG. 8a-8d illustrates a series of steps of the adhesion transfer method according to one aspect of the invention. FIG. 9 graphically illustrates the laser capture microablation technique. Best Aspects for Carrying Out the Invention The invention is directed to a method for analyzing cellular material at the molecular or genetic level, where the method visualizes the field of view of cells in a tissue sample under a microscope. Includes contacting the identified region with a surface that simultaneously dissolves, extracts, and / or maintains the cellular material of interest, and transfers the cellular material of interest to a suitable analytical system. The present invention particularly relates to local tissue polypeptides, proteins such as enzymes and antigens, and DNA. It can be applied to the analysis of RNA, especially mRNA, lipids, carbohydrates, and other biological molecules and their assemblies. According to one aspect, the invention is directed to adherent transfer methods that include microscopic visualization and acquisition of cell material or transfer to a transfer surface. The invention is also directed to a fully automated system, whereby the tissue can be visualized on the screen so that the exact field of view of the cells of interest can be, for example, various labels, histological stains, antibodies. , Especially more identified and limited, or on the other hand, their location can be distinguished and then extracted and analyzed manually or automatically or by a combination of the two means. .. Figure 1 shows how a tissue sample is microscopically imaged, displayed on a display monitor, and selected and analyzed for microexcision and analysis followed by a region of the imaged sample. It is a schematic of a functional system showing a microscope. As shown in FIG. 1, tissue sample 1 is fed onto a surface, eg, a glass slide 2, for microscopic testing and visualization. The sample structure 1 can be immobilized on the glass slide 2 according to any conventional method, for example, binding to the glass slide 2 by an agarose gel, immobilization of the tissue sample on paraffin, and the like. A glass slide 2 with a sample structure 1 immobilized on it is placed on the mounting table of the microscope. The microscope generally indicated by reference number 3 receives an image of tissue sample 1. An imaging device, such as a video camera (not shown), is connected to the microscope 3. The imaging device receives an image of the sample tissue 1 from the microscope 3 and displays the image of the tissue sample on an image display device, such as a display monitor 4. The image of sample tissue 1 is limited to the "field of view" of microscope 3 for any given image. As shown in FIG. 1, the field of view of the sample tissue image stains the cells of interest in the tissue sample, or, on the one hand, the appropriate dye, labeled molecule, eg antibody or its, to identify. By using fragments "C" and "D" can be included. For typical purposes, in FIGS. 1 and 2a-2c, it is assumed that compartment "B" is the compartment of cell material of interest. The image on the display monitor 4 is used by the operator to select and identify one or more sections of interest for tissue sample 1. According to one aspect of the invention, after the compartment of interest has been selected and identified, the operator manually operates the device to extract the identified compartment from the glass slide 2. Identification of cells of interest can also be done automatically through image analysis software. The extracted compartment of the sample material can include the analytical sample. On the other hand, the identified and extracted compartments include compartments to be discarded, and the remaining compartments retained on the glass slide 2 can be analyzed later. In addition to the manual operation discussed in more detail below, according to another aspect of the invention, a microscope that receives a digitized signal of an image from a video camera (or microscope) and holds a sample 3 Using a computer that receives the reference position of the stage, the relative position of the sample compartment is determined. It is possible to use the image on the display monitor 4 to select and identify the sample compartment. It is possible according to one aspect. Such positioning detection and recognition systems are common in the industry and can be readily applied to automate the sample preparation methods of the present invention. In this automated aspect of the invention, the computer for position-fixing detection and recognition is also used to regulate the movement of the devices discussed below used to extract tissue compartments, and thus. Automate sample removal. In addition, the image of the sample can be electronically scanned to automatically identify compartments with the intended features, eg, a reasonable degree of staining, using known techniques and equipment. Thus, in a preferred embodiment, the computer operates such a compartment in order to operate the device to remove the compartment of interest in a fully automated manner. It can be used to select and identify the picture, and the relative position of such compartments. FIG. 2a-2c is a schematic set of functional systems showing how a compartment of tissue sample 1 is extracted from slide-fixed tissue sample 1 according to one aspect of the invention. It should be understood that the steps shown in Figure 2a-2c can be performed manually by the operator or by a computer using conventional position-fixing and control methods, such as a computer-controlled robot. Aspects of the invention shown in FIGS. 2a-2c utilize a contact probe 5 having an adhesive / extraction reagent 6 on its tip. Suitable adhesive / extraction reagents can include a mixture of piccolyte and xylene. In FIG. 2a, the contact probe 5 is manually or computer controlled as described above and is positioned relative to the sample compartment (B) to be extracted. As can be easily understood from FIG. 2a, the surface area of the contact probe tip (and adhesive / extraction reagent) should be approximately equal to or no greater than the surface area of the compartment to be extracted. Otherwise, excessive removal of adjacent tissue compartments will occur. Manufacture of probe tips of the required size is within the ability of one of ordinary skill in the art. When the tip of the contact probe 5 is aligned with respect to the sample compartment (B) to be extracted, the contact probe 5 is lowered so that the adhesive / extraction reagent 6 on the tip is in the sample compartment. Contact with (Fig. 2b). Of course, depending on the characteristics of the device, the probe 5 is lifted or moved to contact the sample compartment of the cell of interest. Adhesive / Extraction Reagent 6 is selected for easy adhesion to the sample compartment. When the adhesive / extraction reagent 6 on the tip of the contact probe 5 comes into contact with the sample compartment (Fig. 2b) and the sample compartment is adhered to it, the contact probe 5 is retracted from the contact position (shown in FIG. 2b). And can be moved as shown in Figure 2c. Since the relative adhesive strength of the adhesive / extraction reagent is greater than the adhesive strength used to secure the sample onto the glass slide, the contact probe 5 will be removed from the glass slide when it is pulled out or retracted from the sample compartment B. Attract. According to one aspect of the invention, a glass pipette was used as the contact probe 5. In this embodiment, the tip of the glass pipette was coated with a solution of picolite (568 g / l) and xylene (437.5 g / l) by impregnating the tip of the glass pipette with a solution of picolite / xylene. In addition to removing the sample compartment from the glass slide 2, the contact probe 5 places the sample compartment extracted in the analysis vessel 7 or in any other location, such as a waste vessel, culture medium, etc., as shown in FIG. 2c. Can be used to transport. In a preferred embodiment, the contact probe 5 is used to transfer the extracted sample compartment to the sample acceptance stage of an automated clinical analyzer designed to perform the desired analysis of the sample compartment. Thus, the present invention identifies a sample compartment on a surface, eg, a sample on a slide, removes a sample compartment of interest from a surface-fixed sample, and automatically removes the extracted sample compartment from that extracted sample compartment. It should be understood that it is possible to provide fully automated methods and systems for transport to automated analyzers capable of performing the analysis. Such analysis includes, for example, cellular DNA, Includes analysis of RNA, proteins, polypeptides, lipids, carbohydrates, and combinations and aggregates thereof. In FIG. 2c, the extracted sample compartment is shown to be dispersed in a test tube or container 7, which can be a similar vessel, on which analysis of the extracted sample compartment can be initiated or performed. As shown in FIG. 2c, reagent solution 8 for removing all or desired components of the extracted sample compartment from the tip of the contact probe is placed in container 7 before the extracted sample compartment is provided there. Can be done. For example, for DNA analysis, Tris (50 mM, pH 8.5), EDTA (1 mM), Tween 20 (0.5%) and Proteinase K (0. 2 mg / ml) solution can be used. This solution extracts a sample compartment from the tip of the contact probe 5 and dissolves the tissue material for analytical purposes. In addition to the contact probe shown in Figure 2a-2c, a hollow suction probe can also be used to extract a sample compartment from a slide-fixed tissue sample. Such a suction probe may have a sharp annular tip from which the sample compartment is aspirated and extracted by a suction force. FIG. 3 is an illustration of another device for extracting sample compartments from slide-fixed tissue sample 1. The extraction device 9 shown in FIG. 3 has a cutting blade 10 and a capture arm ll. The capture arm 11 can be moved in the opposite manner to the cutting blade 10. The capture arm 11 is shown in its open position in FIG. The capture arm 11 can move from a closed position where the tip of the capture arm 11 contacts the cutting blade 10 to an exemplary open position. The movement of the capture arm 11 can be regulated by a cable and pulley system that causes rotation at the base of the capture arm 11 by pulling a cable that passes through a pulley located at the base of the capture arm. Tension on the cable can be applied by activating a lever on a device that applies tension to the cable in a known manner, or by pressing a button 12. Such actuating mechanism structures are known in the gripping device industry. In the operation of the device of FIG. 3, the cutting blade 10 present at an obtuse angle with respect to the central axis of the device places the cutting blade 10 on one end of a portion of the tissue sample to be extracted, and then In addition, by moving the capture arm 11 to the closed position, a portion of the tissue sample can be cut and scooped up. As the capture arm 11 comes into contact with the tissue sample, it draws the cutting blade 10 into the sample and pushes a portion of the sample towards the cutting blade 10, thereby causing the cutting blade 10 and the capture arm 11 to come into contact with each other. Causes cutting and scooping of some of the samples that are in contact with each other. Figure 3 Further of the device In another embodiment, the movement of the capture arm 11 can be provided by gears instead of pulleys and cooperating dentate rods instead of cables. Additional such mechanical structures are known in the capture equipment industry. 4a and 4b are schematics of a manual extraction tool manipulator that can be used with the extraction device of FIG. 3 according to the present invention. In FIG. 4a, the extraction tool manipulator has a base 13 with a tightening means 14 for detachable coupling of the device to the brace or support of the microscope platform (see FIG. 4b). It is depicted as a thing. The tightening mechanism includes a tightening plate 15 fixed to a screwing shaft 16 passing through a screwing cavity 17 in the lower portion of the base 13. A tightening knob 18 is fed over the end of the screw-in shaft 16. The rotation of the tightening knob 18 causes the tightening plate 15 to be removed with respect to the upper portion 19 of the base 13. Therefore, the extraction tool manipulator positions the brace or support portion 21 of the mounting table of the microscope 20 between the tightening plate 15 and the upper portion 19 of the base 13, and the brace or support portion of the mounting table of the microscope 20. By rotating the knob 18 to connect the tightening plate 15 to 21, it can be tightened to the portion of the mounting table of the microscope 20 as shown in FIG. 4b. The extraction tool manipulator includes a tool holder 22 having a through cavity 23 therein for receiving the extraction tool shaft 24. Ideally, the tool holder 22 should allow the extraction tool to move back and forth in a braked manner. Thus, according to a preferred embodiment, the through-cavity 23 of the tool holder 22 includes a bush that can be adjustablely fastened to the tool shaft by a tool 24 lacking a screw. The tool holder 22 is supported by a support shaft 25 connected at an end opposite the swivel 26 and 27 that are substantially damped to the tool holder 22 and the base 13. The length of the support shaft 25 between the swivels 26 and 27 that are braked 360 degrees is adjustable. Support shaft Independent 360 degree braking swivel 26 and 27 adjustments, along with 25 adjustable lengths and the position of the tool axis within the through cavity 23, slide-fixed samples placed on the microscope platform. On the other hand, it enables a high degree of movement of the extraction tool. Thus, the operator can operate the extraction tool maintained by the extraction tool manipulator and move the tissue compartment selected from the slide-fixed tissue sample with a high degree of accuracy. FIG. 5 is a schematic functional system diagram showing how the sample tissue compartments can be directed to the appropriate analytical protocol. As shown in FIG. 5, microextraction of tissue sample compartments was taken from slide-fixed tissue sample 1 as discussed above, and cells of interest were extracted for analysis. It can then be transferred to sample preparation step 28 to be harvested. The extracted cells can also be lysed at this stage. If those cells contain or are likely to contain one or more DNAs or RNAs of interest, the sample extracted will be amplified by polymerase chain reaction (PCR), followed by, for example, hybridization, stranded polymorphisms. It can be left to the phenomenon, Southern and Northern plots, sequencing, etc., if desired. Of course, other techniques for the analysis of DNA and RNA are known to those of skill in the art and are covered by the scope of the invention. If the extracted cells contain or are likely to contain a protein or polypeptide of interest, the extracted sample will be, for example, an enzyme zymography using one or more labeled substrates, labeled antibodies. Or it can be entrusted to immunoassays, biochemical assays and similar means using its functional fragments. Selective extraction or microexcision of frozen tissue fragments according to the present invention allows for the recovery and analysis of active enzymes and mRNA. In addition, the DNA recovered from those fragments exists under native conditions and can be used for research, such as DNA fingerprinting. Microexcision of paraffin-embedded tissue according to the present invention is 1 Allows PCR amplification of DNA from a pure cell population that exhibits less than one high field of view, or a single layer of epithelial cell inner follicles space. For general preparation of samples for frozen fragment microscission according to the present invention, microscission slides can be prepared by placing 1% agarose and coverslip on standard histological slides. .. After a short period of time, eg about 5 minutes, the coverslip is removed leaving a thin gel on the slide. A small frozen tissue fragment, for example about 25 microns thick, is placed on an agarose gel and immediately stained with eosin. Tissues can also be treated with agents to denature or, on the other hand, inhibit RNase, depending on the subsequent extraction method. Under direct microscopic visualization, a particular cell population or subpopulation of interest is acquired from the tissue fragment using the techniques discussed above. For enzyme analysis, the obtained tissue specimen can be placed in a suitable buffer depending on the enzyme of interest, as known to those of skill in the art. Enzyme levels can be measured by several methods, such as zymography, and by the use of specific substrates, such as fluorescence measurements, colorimetric measurements and radioactive substrates. Thus, the exact level of enzyme expression in a particular predefined cell population can be determined and, if desired, compared to the level of another independently isolated sample from a tissue sample. For mRNA analysis, tissue specimens can be placed on agarose and, optionally, treated with agents to denature RNase or, on the other hand, inhibit it. The obtained tissue specimen is immediately frozen under liquid nitrogen. Tissues can be used immediately or stored at -70 ° C for several months. mRNA can be extracted using, for example, column chromatography on oligo-dT (Micro-Fast Track mRNA Isolation Kit, Invitrogen Co. ). The recovered mRNA of a pure cell population can also be amplified and investigated using polymerase chain reaction (PCR) by RT-PCR as known to those of skill in the art. For DNA analysis, tissue samples were placed in a single-step extraction buffer solution of 50 mM Tris, pH 8.5, 1 mM EDTA, 0.5% Tween 20 and 0.2 mg / ml Proteinase K at 37 ° C. It can be incubated for 4 hours, followed by 10 minutes at about 95 ° C. The recovered DNA can also be amplified and analyzed using PCR techniques in combination with analytical techniques known in the art such as blotting, sequencing, etc. If native DNA is required for DNA fingerprint analysis, proteinase K can be added after DNase in the fingerprint protocol. For paraffin fragment microexcision, conventional formalin-fixed, paraffin-embedded tissue sections are microexcised after paraffin removal and immediately stained with eosin. Tissue fragments are visualized by direct microscopy and the cell population or subpopulation of interest is acquired using an improved glass pipette with the adhesive coated tip discussed above. A tissue sample as small as one cell is obtained by this method. The peculiarity of cultivating shows significant improvement over currently known techniques. For DNA analysis of paraffin-embedded tissue, a glass pipette containing the excised tissue specimen removes tissue from the tip of the pipette, 50 mM Tris, pH 8.5, 1 mM EDTA, 0.5% Tween 20 And 0. Placed in a single-step extraction buffer solution of proteinase K at 2 mg / ml. Samples are incubated at about 37 ° C for 2-24 hours, followed by about 95 ° C for 10 minutes, depending on the sample size. The tip of the glass pipette is then sterilized and reused, but this is generally not recommended for PCR-based minutes due to the potential amplification of cross-contamination materials. In another aspect of the invention, one or more cells of interest are isolated by laser capture microexcision as illustrated below. The main operation of laser capture microexcision is shown in Figure 9 (microscope not shown). In this method of the invention, the tissue sample sample is immobilized on the support as described above, and a transparent or translucent film or tape (transfer film) is placed on top of the tissue sample sample. Tissue samples are then microscopically tested on the intended target cells, such as abnormal cells (or control cells for comparison). As mentioned above, cells can be stained by dye, immunologically, etc. to identify and / or differentiate the cells of interest in the sample. The cells of interest are then made to coincide with the target point where the electromagnetic rays are concentrated. This match can be achieved, for example, by xyz translation of either the sample or the target point. For example, the target point coincides with the center of the image field and microscope platform that is translated so that the cells of interest are brought to this target point. The energy of one or more concentrated pulses (eg, electromagnetic energy in the form of light from an infrared laser, thermal energy, etc.) is directed at the film covering the target. Sufficient energy is selectively heated at the film or tape covering the cells of interest at the target point, or on the other hand, directed at the target point to alter their adhesive properties. In this case, the film or tape is selectively adhered to a particular target in the sample by optical activation at exactly a predefined position. Preferably, the laser is used in the present invention to supply electromagnetic energy to the target spot. This is because the laser is a very bright light source of strong parallel light that can be easily and effectively focused on a small area on a constant surface. By laser focusing on the optical center of the field of view of the light microscope, activation energy can be concentratedly delivered to the target area of the film or tape located above the tissue sample. In addition, the timing and duration of the laser can be readily adjusted so that a regulated amount of energy can be directed to the target point. In addition, the laser beam is focused on spots as small as the diffraction limit of the wavelength used, and thus allows selective adhesion to targets as small as 1 micron. Therefore, the spots are small enough to select cells of homogeneous clusters, individual cells, or even parts of cells. The adhesive layer specifically targets the film or tape when sufficient energy from the focused radiation pulse is absorbed to provide activation of the film surface in contact with the cells of interest in the tissue sample. It is formed with the cells that have been formed. As long as the focal bond strength formed between the film and the targeted tissue is greater than the bond strength of the targeted tissue to the underlying support (eg, a microscope slide), the targeted tissue will Can be obtained based on film removal. Regions of the unactivated film form weaker bonds with untargeted regions of the sample tissue slice than the strength of particles (eg, intercellular) within the tissue sample, and those intercellular connections form the basis. If the bond between the tissue and the underlying support is weaker than the bond formed between the activated film and the targeted tissue, then the target The formed tissue selectively binds to the film or tape and can be selectively removed if the film is stripped or removed from the tissue slide. Depending on the needs of the operator, the size of the tissue to be transferred depends on the laser beam It can be changed by changing the diameter of the pulse and the duration of the pulse. High regenerative transport in the 60-700 μm diameter range can easily achieve the acquisition of small (100 μm-1 μm) lesions without invading adjacent non-neoplastic cells. In the most basic and clinical studies, the acquisition of hundreds to thousands of cells is required to provide sufficient genetic material for reliable amplification and statistically meaningful analysis. .. However, since the laser beam can be concentrated smaller than the diameter of one cell, it appears that transfer of a single targeted cell or even a portion thereof is possible in the practice of the present invention. Thermoplastic polymer films are widely used as heat and pressure activated adhesives for bonding surfaces. Most of those polymer films without the added dye are transparent or translucent to the visible light used in conventional light microscopy. However, these films are found in certain regions of the electromagnetic spectrum (eg, in the infrared region associated with strong molecular vibrational modes, eg 3000,1800,1400-960 cm).^-1), It is inherently strongly absorbent. It is also possible to add infrared absorbing dyes to the thermoplastic film to provide strong absorbency at other specific infrared wavelengths without changing their transparency to visible light. Such dyes are preferably IR-absorbing dyes such as metallonafter Russianin, naphther Russianin, and cyanine dyes. If a focused pulse of electromagnetic rays (eg, a laser) is supplied at a wavelength that is strongly absorbed by the film, the film can be rapidly and effectively concentrated and heated. The indicator can also be included in the selective adhesive transfer film or in separate layers to define the location of the optical activation. Such indicators are converted to colorants by thermochromic dyes, dye precursors that bind based on melting to form colors for visible or instrumental identification, and other effects of optical absorbency. Includes dyes. Suitable indicators also include the appearance or disappearance of transparency or opacity based on physical effects such as optical exposure or heating. Without being bound by theory, if such thermoplastic films are heated near or at the melting point, they flow to adjacent surfaces (in this case, the targeted tissue sample), and It seems to fit and form a strong surface bond. This binding appears to occur without actual chemical cross-linking to the tissue sample. Such strong bonds are most reliably formed when pressure is applied to force a "melt" flow to a strong compatibility with the sample surface. However, due to the high reproducible intensive microtransfer that occurs by using a smooth film that is applied in close proximity to the tissue and by supplying the appropriate pulse parameters to the thermoplastic film of the selected composition. The film can be reliably and intensively heated to the peak temperature associated with high film fluidity for a sufficient period of time to form a properly strong bond between the film and the tissue. In addition, a pulsed infrared laser source is used to activate the intensive binding of the targeted tissue to the film. By doing so, the targeted tissue retains the tissue morphology of focus and is quantified without chemical denaturation, allowing unaltered microscopic observation before, during, and after microtransfer. Acquired (substantially complete microtransfer to film). Any additional steps also improve the binding between the cells of interest and the activated polymer film and reduce the binding of the tissue of interest to the support, resulting in easier selected cells. It can be used for laser capture microablation so that it can be removed. The slides can be selected to be (i) a material with a inherently lower affinity for the tissue sample than the polymeric film, (ii) pretreated with an agent that reduces this affinity, or (iii) melted. It can be encapsulated in a material that is compatible with the resulting polymeric film. For example, glass slides with tissue on it can be processed according to conventional slide preparation methods by impregnating with a 3% aqueous glycerol solution and then drying. On the other hand, the structure can be encapsulated in a polymeric material that forms a strong bond with the meltable film and is sufficiently water soluble to allow recovery of the tissue sample during the analytical phase. Encapsulation of the tissue in such a material can be done by a coating technique, eg, application of a polymer to a solution, or by placing the material in film form on the structure and melting it. Encapsulation can also be present in the form of coaching on the hot melt film to strengthen the bond of the meltable film to the tissue and reduce its bond to the slide. In addition, it strengthens the bond strength between the tissue and the targeted surface, reduces the bond strength between the tissue and the slide, and allows reliable removal of the tissue / molten polymer from the slide. Depending on the material, it is possible to process surface / texture combinations. Any wavelength of electromagnetic energy can be used under the practice of the present invention under the condition that suitable materials are used. In particular, the transfer film melts or nearly melts the thermoplastic polymer in the targeted area. It is important to absorb enough energy (or include one or more dyes to absorb enough energy) at the wavelength chosen to do so. For thermoplastic materials such as ethylene vinyl acetate, wavelengths of about 3 to about 10 μm are preferred as they naturally absorb in this range. The power of the laser used generally depends on the size of the target (ie, the power increases as the size of the target increases), ranging from about 1 mW to about 200 mW, preferably about 10 mW to 100 mW. Exists in. It is also preferred that the wavelengths for laser activation and film absorption be selected outside the normal range used for microscopic images. Renewable microtransfer of tissue is obtained using a variety of infrared wavelengths (9.6-11 μm) from a tunable carbon dioxide laser. The transfer film can be selectively activated by electromagnetic or thermal energy and is preferably either transparent or translucent with respect to the visualization wavelength. This selectively activating transfer film can be made, for example, from a wide variety of thermoplastic materials such as ethylene vinyl acetate, polyurethane, polyvinyl acetate, and the like. In one aspect of the invention, the selective activation transfer film is a film of a polymerizable substance, eg, an electromagnetically activated polymerizable substance that polymerizes based on exposure to electromagnetic energy. Certain other selective activating materials found to be useful in the practice of the present invention are: thermal adhesives and waxes, Precision Coatings product # HAL-2 180C; thermally activated Thermal adhesives and sealants, such as from Ban Fastening Systems (Brooklyn, NY); UV sensitive or curable optical adhesives, such as ThorLabs, Inc. Product NO60-NOA81; ). The adhesive film can be a self-supporting film or can be laminated by a support film. In addition, the supporting film can be made from a material that does not absorb electromagnetic energy so strongly that it substantially interferes with the activation of the thermoplastic polymer. The support preferably absorbs weakly at the activation wavelength and at the visualization wavelength. On the other hand, the activable film preferably absorbs weakly at the visualization wavelength, but strongly at the activation wavelength. The support should also not be affected by the resulting thermal transfer that occurs during activation. The use of microscope slides and clear tape is preferred in practicing one aspect of the invention. Observation of slides under a microscope means that a pathologist or other microscopic observer selects one or more spots on a tissue sample and exposes them to (invisible) infrared energy from any suitable optical system. To enable. The film becomes adhesive at one or more selected spots, and the tissue at those spots adheres easily to the polymer film, which is then removed from the slide to hold the tissue sample with the film. To. The selected sample is then followed by the desired analysis, especially altered gene expression as measured by gene mutation / deletion, mRNA and / or protein concentration, changes in enzyme activity, and molecular analysis of similar ones. In addition, it can be removed from the film by appropriate techniques. Since one or more samples of cells of interest are obtained from a single sample slice, normal cells are also obtained from the same tissue and analyzed molecularly, and analysis of cells of interest along with normal cells is useful diagnostics and Can be compared for prognostic information. Laser microablation systems can be conveniently used for a variety of sample preparations, such as stained slices of tissue or stained cytological specimens of intact cells. In this case, the transferred area can be clearly identified microscopically by the intensively transferred, stained material on a clear film. On the other hand, films and tissue slides can subsequently be recorded automatically. It may be displayed in the xy coordinate system to align a particular slide position to an individual transferred point. The micro-transferred tissue is then micro-transferred, for example, by directly punching a spot accurately recorded in the desired reaction or extraction vessel (eg, by automatic xy translation), or the entire film into the reaction vessel. Can be collected from the film by arranging. In one embodiment of the invention, molecular analysis of the extracted cellular material, eg, by RT-PCR, is to localize small objects (eg, 50 μm spot tissue) that adhere to the support, and strike into the vial. Requires their collection into a extracted analysis chamber. The location of individual target sites and the use of automated translational xy cords have the same coordinates of the region to be punched, unless the supporting film is deformed or stretched during the application, activation and removal steps of the process. Allows automation using. Therefore, the sample collection process is also sensitive to automation. On the other hand, it can be ensured that the target site is present at a known location on the transfer support. For example, multiple pieces of adhesive transfer film are selectively applied to their location on the tissue corresponding to the site to be extracted, rather than applying a single large piece of supporting film to the complete specimen. Can be done. A typical such chem is constant on a continuous polyester film, preferably a strong and not easily stretched sheet, to provide a linear array of target sites where a small disc of adhesive film can be activated separately. It is applied at the repetition distance of. After the individual target compartments have been identified, the next unused small adhesive spot in the linear array is locally applied at an isolation distance fixed to this area by a small pressure plate or air jet. The support / tissue slide as a unit then targets specific cells (determined by the laser spot diameter) within the target compartment (ie, the diameter of the adhesive spot, which is larger than the diameter of the laser spot). The fine position is determined under microscopic observation. To. When the support film and the device applying those small pieces of film are located relative to a fixed control (eg, a microscope objective), the adherent tissue spots are always present on the support film (eg, the adhesive film at equal spatial distances). If so, it is supposed to be in a known position on (in the central part of the narrow strip). Targeting of similar cells within the target compartment is achieved by microtranslating the sample between successive laser pulses. Thus, the operator positions the slide in the microscope so that the tissue of interest is in the center of the field of view, and then a pressure plate or other means, such as an air jet, is the cell / object of interest. Adhesive film spots on the tissue can be juxtaposed so that is within a known position on the support film. The adhesive is activated by an IR laser pulse, and then the pressure plate is opened. The film is then separated from the specimen slide and the fresh portion of the film is advanced so that it can be used in the next specimen position. The process allows the occurrence of surjective transfer onto a series of regular (numbered array) spots with fixed conformation, as the film is not deformed in those processes. The size of the adhesive film spot determines the size of the target compartment, within which the object / cell selection is made for that one transfer (array number) in aspects of the invention. The size of the target compartment is determined by the choice of a particular film (ie, the geometry of the regular array), but can be increased or decreased by different sized parallel rows of spots on the same supporting film. Can be done. An example of an adhesive transfer film is a small piece with or without a carrier support, or an isolated portion of the adhesive film, preferably an equally spaced portion. In this regard, the use of tape provides the ease of transfer of a new portion of the film to the working area and the ease of removal of the activated film into a collection or storage means. Adhesive fee from tissue Removal of the rum can be achieved by pulling the tape while keeping the support (eg, slide) in place. This is achieved in aspects of the invention using a pressure plate after the pressure plate has been removed. This is often the mechanical device for tape transport, and tension, where the adhesive film maintains tape alignment during the tension and pressure plate actuation processes (eg, symmetrical deflection in the lateral tape transport direction). It needs to be tightly bound to a support that will not change below. In another further simplified aspect of the invention, the collection process is carried out by cutting (rather than punching) the desired portion of the adhesive film. This simplified aspect eliminates the requirement for close resistance between the punch and die. On the other hand, other means can be used to separate the tissue / adhesive film from the rest of the tape. Such means will be apparent to those of skill in the art based on the disclosure herein, and direct peeling of the tape, focal dissolution of any of its bindings to the adhesive tape, or supporting film, heat ray knives (between specimens). Includes spot removal by self-sterilization) to eliminate contaminants. A further feature of the invention is directed to sample identification. Small pieces of film can have tiny identification marks (eg, barcodes) attached to those small parts that can be found under a microscope and recorded with a video image of the specimen. A practical means of achieving this is to place identification marks on mechanically strong supports adjacent to individual separate spots of adhesive based on the tape morphology described above. Further use of those identification marks is to regulate the progression of the tape for individual new specimens. A microscope, or a sensor in the analysis of various specimens, determines when the mark is present, for example, in the center of the field of view as the tape is advanced, and then stops tape transport. On the other hand, mechanical drives (eg sprockets) Has), but a fixed known amount, can be used to advance the tape. In a preferred embodiment of the invention, equally spaced adhesive film spots along the microadjacent bar code discriminator are thin (eg, 1 mm) wide, mechanically strong backing tape, eg, preferably a reader. Supplied to a sterile cassette with about 0. Located in the center of a 002 inch thick Mylar. The cassette and stepper motor take-up drive is coupled to the housing of the reversing microscope (or its stationary stage if the slide is to be moved manually) so that the center of the tape is centered on the microscope objective (and the microscope objective). Aligned with the center of the field of view), and the tape is present above the sample level. The leader is coupled to the spool on the drive shaft of the stepper motor and is fully wound, so that the tape is tightly coupled to the winding motor and the first adhesive spot is in the correct position as planned. The solenoid working pressure plate is pushed down onto the tape so that it is close enough to the specimen and the sensor (video signal) in the microscope is tightly bound to the specimen so that the tape can be advanced to its exact final position. The identification mark is then advanced to its final position, and the pressure plate is pressed firmly against the film. The IR laser is activated to bind the selected tissue to the adhesive film, and shortly thereafter, the pressure plate is released. The pressure plate solenoid, which holds and holds the two rakes lying between the film and the platform, is then temporarily activated upwards so that the two rakes remove the tape from the tissue. Will be done. The next sample is optionally selected by the operator, and the pressure plate is activated partially below it so that the sensor can detect the identification mark, and the process is repeated if desired. After the final specimen has been transferred, the motor advances the tape further, and then the take-up spool and cassette are removed and coupled to the motor shaft of the collector fixed on the loading platform. This device uses a heat ray knife to cut out adhesive spots / adherent tissue from the rest of the tape, and separates the two (if necessary), and samples either a vial or a 96-well microtiter plate. Use an air jet to deposit. Adhesives along the length of the tape Known positions in the pot can be used to accurately position the first spot and advance the tape to subsequent positions. Computer-generated barcodes that correlate with film labels are attached to vials or microtiter plates for tracking. The barcode is stored in the computer data entry of the microtransfer sample (eg, image, number of patients, number of samples, etc.) and is recorded directly on the image of the target immediately after laser activation. Laser capture microablation (LCM) has many advantages over prior art: LCM is (1) easy to carry out, (2) in its simple form, does not require moving parts, (3) does not require manual dexterity or manipulation, and is most important. In particular, (4) transfer is a one-step process. In addition, the tissue transferred to the film retains its morphology, allowing microscopic verification of the specificity of the captured material prior to molecular analysis. A further advantage of the present invention is that the use of sterile disposable film for transfer minimizes any possible contamination problems that are of particular importance in analysis using PCR techniques. Yet another advantage of the present invention is that the capture film is sufficient for a small amount of energy, a small, inexpensive, low power laser (50 mW or less) that can be combined with a standard microscope to provide complete transfer. Can be activated by such energy. As an example of the advantages of the present invention, individual glomeruli are captured from a kidney tissue fragment sample in less than 10 seconds, and hundreds of glomeruli are isolated by one operator in an hour with minimal effort. obtain. Those skilled in the art will appreciate that such speed and efficiency cannot be approached by conventional microcutting methods. It should also be understood that laser capture microexcision is not limited to use on biological samples. In fact, the techniques described herein can be used to classify / remove any object that needs to be distinguished from other objects in the microscopic field of view. For example, micromachined objects can be easily, rapidly and effectively classified under the practice of the present invention. It is further understood that the practice of the present invention is not necessarily limited to the use of electromagnetic energy such that any energy source that provides a particular localized melting of the thermoplastic transfer film would operate in the present invention. Should be. A heat source from the electrical circuit is desired when the area to be transferred is large enough, such as in a relatively homogeneous tissue sample with a size of approximately 1 mm. Electrically heated radiation beaters, as will be apparent to those skilled in the art based on the disclosure herein. (As obtained from CA), and the like, are also useful as sources of selective energy. The properties and features of the invention are illustrated by the following examples, but they do not limit the invention. In the example, the percentage is by weight unless otherwise stated. The following examples were carried out in an attempt to establish whether the present invention is used to study protease distribution more specifically during human tumor invasion. Measured to assess whether MMP-2 and catepsin B levels in the field of invasive breast and colon cancer were quantitatively elevated relative to the number of normal cells from the same patient in those areas. Was done. In the following example, normal and tumor samples of colon and breast tissue from surgical resection were maintained frozen (-70 ° C) until analysis. Tissue fragments of invasive breast and colon cancer were selected based on histological evaluation. For tumor fragments, histological regions of tissue containing invasive tumors and substrates were selected, except for significant numbers of normal epithelium or sputum cells. A control fragment of normal tissue contained an epithelium and a thin fragment of the underlying substrate. The proportions of epithelial and substrate tissue were similar for normal and tumor fragments. In an example, microexcised slides were prepared by coating a standard histological slide with 200 μl warm agarose (1%) and then covering it with a coverslip. After 5 minutes, the coverslip was removed, leaving a thin layer of agarose on the slide. Frozen fragments with a thickness of 20 microns were prepared in a cryostat and placed on an agarose gel. The tissue was briefly impregnated with eosin. Optimal microexcision was achieved by starting at the edges of the individual fragments and systematically cultivating and separating the histological part of interest with the microexcision device of FIG. The part of interest was retained on the slide for subsequent analysis. The DNA content in the sample was determined by spectrophotometric measurements at 260 nm. DNA content in individual samples is measured in individual histological fragments It was proportional to the number of cells. A cDNA (and DNA) library of microexcised tissue fragments is also provided by the present invention and the methods of making such libraries. Such libraries are useful in their own right in facilitating the identification of transcripts that are specifically expressed in cells of clear histological origin and at the stage of tumorigenesis.<u style="single">Example 1</u>In this example, samples of normal and tumor tissue adapted for cell count were analyzed from individual subjects. Levels of MMP-2 were determined by zymography and quantified using an Arcus scanner. The results were statistically analyzed using Student's t-test. Cathepsin B level, V against substrate Z-Arg-Arg-NHMec_maxWas decided as. The results of this example are shown in Table 1 below, which lists cathepsin B activity in matched pairs of invasive colon cancer / normal epithelium and invasive breast cancer / normal epithelium. The activity measurement value is V_max, N mol / min x mg DNA. Cathepsin B activity was increased by an average of 2.3-fold (p &lt;0.005) in colon cancer and an average of 6.9-fold (p = 0.077) in breast cancer.<img file="JP3958366B2_D0001.tif" />As can be seen from Table 1, 9 of all 5 breast cancers and 10 colon cancers showed increased activity of cathepsin B compared to the number of normal cells from the same patient. (table 1). The enhanced activity in colon cancer ranged from 19% to 283%, and the average increase in tumors was more than doubled. The increase in cathepsin B activity was more pronounced in breast cancer, and the average increase was slightly less than 7-fold.<u style="single">Example 2</u>In this example, polymerase chain reaction (PCR) analysis was performed. Based on the previously reported cDNA sequence of 72KDa type IV collagenase, sense and antisense oligonucleotide primers were synthesized for amplification of the enzyme activation site (such as M. Onista, "Reverse Transcription-Polymerase Chain Reaction"). Phenotyping of Metalloproteinases and Inhibitors in Tumor Matrix Invasion , Diagn.Mol.Pathol.2 (2): 74-80,1993), the paired oligonucleotide sequences were: 5'-CAA TAC CTG AAC ACC TTC TA, 3'-CTG TAT GTG ATC TGG TTC TTG. Labeled PCR for single-stranded conformational polymorphism (SSCP) was obtained by combining the following in a 10 μl reaction: 1 μl of 10 × PCR buffer (100 mM Tris-HCl, pH 8.3; 500 mM KCl; 15 mM MgCl₂0.1% w / v gelatin); 1 μl of DNA extraction buffer; 50 p mol of individual primers; 20 n mol of individual dCTP, dGTP, dTTT and dATP; 0.2 μl of [³²P] dCTP (6000 Ci / m mol); and 0.1 units of Taq DNA polymerase. The amplification reaction was carried out at 95 ° C for 30 seconds, at 60 ° C for 30 seconds, and at 72 ° C for 30 seconds for 30 cycles. FIG. 6a shows the expression of MMP-2 in 10 invasive colon cancer cases compared to the normal colonic mucosa from the same patient. The bar graph shows about a 3-fold increase (p &lt;0.001) in the pre-form of the enzyme 72KDa and a 10-fold increase (p &lt;0.001) in the active form of the enzyme 62KDa. Figure 6b shows the expression of MMP-2 in 5 cases of invasive breast cancer. The bar graph shows a 3-fold appropriate rate of increase (p &lt;0.05) in the 72 KDa preform of the enzyme and a 10-fold appropriate rate of increase (p &lt;0.05) in the active form of the enzyme 62 KDa. The 62KDa active form of 72KDa protype IV collagenase and the enzyme was enhanced in all 10 colon cancers and all 5 breast cancers compared to normal tissue from the same patient. The rate of increase was high in the 62KDa active form of the enzyme, which was increased by an average of 10-fold in both colonic and breast cancers compared to normal control tissue. Proenzyme levels at 72KDa were increased by an average of 3-fold in both tumor types. For both breast and colon cancers, the elevation of 62KDa in reactive enzymes was more variable than the rate of elevation in proenzymes. The rate of increase in 62 KDa in active enzymes in tumors ranged from 3 to 20 times, and the rate of increase in 72 KDa in proenzymes consistently ranged from 2 to 5 times. These results include Davis et al. (Activity of Type IV Collagenases in Benign and Malignant Breast Disease, Br.J.Cancer, 67: 1126-1131, Similar to the recent findings in the analysis of human breast cancer in 1993). The authors performed a zymogram analysis of tissue fragments from human breast cancer patients. Their analysis shows that the total MMP-2 fraction present as an activated form of 62KDa is statistically enhanced in malignancies, and a high proportion of this reactive oxygen species is detected in tumors to a higher degree. I showed that. The present invention extends this analysis by comparing and quantifying both 72KDa and 62KDa forms of enzymes in specific areas of invasive tumors and normal control epithelium from the same patient.<u style="single">Example 3</u>In this example, a single-strand conformational polymorphism (SSCP) analysis was performed. The labeled and amplified DNA was mixed with an equal volume of formamide-filled dye (95% formamide; 20 mM EDTA; 0.05% bromophenol blue; and 0.05% xylene cyanol). Samples were denatured at 95 ° C. for 5 minutes and loaded onto a gel consisting of 6% acrylamide (49: 1 acrylamide: bis), 5% glycerol and 0.6 × TBE. Samples were electrophoresed at 8 W at room temperature overnight. Transfer the gel to 3 mm Whatman paper, let it dry, and perform autoradiography in Kodak. This was done with X-OMAT film. Figure 7 shows SSCP analysis of the MMP-2 activation site. This figure shows a representative case of normal colonic mucosa compared to invasive colon cancer and a representative case of normal breast tissue compared to invasive breast cancer. No differences between normal and tumor specimens are observed. The two bands in the individual line represent single-stranded and double-stranded DNA. Similar results were obtained for 10 colon cancers and 4 breast cancers. DNA encoding the activation site of gelatinase A from colon cancer and breast cancer using PCR to assess whether the elevated tumor levels of activated MMP-2 are due to mutations in the enzyme. The sequence was amplified. Its activation site is located 10 KDa from the N-terminus of the enzyme and contains a cleavage site that converts 72 KDa proenzymes to 62 KDa active species. Amplification and analysis of this region by PCR and SSCP showed no detectable mutations in any of the 10 colon cancers or 4 breast cancers studied. These results suggest that elevated levels of reactive enzymes in invasive tumors are most likely due to tumor-associated activated species. It was determined that the sensitivity of PCR amplification of DNA from microexcised frozen tissue fragments was lower than in one high magnification field of view. Similar to DNA amplification, mRNA amplification from a small cell population was performed according to the present invention using reverse PCR. Previous studies have shown that MMP-2 is up-regulated in human colon cancer. However, some recent studies using field hybridization analysis have reported that MMP-2 mRNA levels in human colon cancer are elevated in substrate cells as opposed to tumor cells. To address this possibility, frozen tissue fragments were microexcised and enzyme levels of MMP-2 in separate tumor and substrate cell populations were measured. Sufficient tissue was harvested from a single high magnification field of view and enzyme levels were quantified by zymography. Invasive tumor cells and adjacent in 3 cases Substrate studies show that 72KDa bro-MMP-2 and active 62KDa forms are associated with both tumor cells and substrate cell populations. Preliminary data suggest that the highest enzyme levels are present at the tumor-substrate interface. According to a preferred embodiment, the invention can be directed to adhesive transfer methods that include microscopic visualization and transfer of cellular material to the acquisition or transfer surface. According to a general method, the adhesive surface is placed in contact with the surface of the cell or tissue, and the adhesive force binds the cell material of interest to the adhesive surface. An adhesive surface, which can be the tip of a tool or needle, is used to acquire the material and transfer it to a liquid analysis reaction mixture. Examples of adhesive surfaces include the use of an adhesive coating on the tip of a tool, or the electrostatic force between the tip and the surface of the cellular material. As described in detail below, the isolation and transfer methods of the present invention are specialized continuously active adhesives applied to cellular materials on regions larger than the region selected for microscopic acquisition. It can include layers or surfaces. According to a preferred embodiment, a laser or other electromagnetic source is used to activate the adhesive force between the cell material and the active adhesive layer or surface. This allows the accurate generation of adhesive force only in the exact microscopic area selected. A suitable laser provides a wavelength that is absorbed, preferably more strongly absorbed by the film. Such a laser is CO Used for transport. Examples of adhesive surfaces include the use of an adhesive coating on the tip of a tool, or the electrostatic force between the tip and the surface of the cellular material. As described in detail below, the isolation and transfer methods of the present invention are specialized continuously active adhesives applied to cellular materials on regions larger than the region selected for microscopic acquisition. It can include layers or surfaces. According to a preferred embodiment, a laser or other electromagnetic source is used to activate the adhesive force between the cell material and the active adhesive layer or surface. This allows the accurate generation of adhesive force only in the exact microscopic area selected. A suitable laser provides a wavelength that is absorbed, preferably more strongly absorbed by the film. Such a laser is CO Used for transport. Examples of adhesive surfaces include the use of an adhesive coating on the tip of a tool, or the electrostatic force between the tip and the surface of the cellular material. As described in detail below, the isolation and transfer methods of the present invention are specialized continuously active adhesives applied to cellular materials on regions larger than the region selected for microscopic acquisition. It can include layers or surfaces. According to a preferred embodiment, a laser or other electromagnetic source is used to activate the adhesive force between the cell material and the active adhesive layer or surface. This allows the accurate generation of adhesive force only in the exact microscopic area selected. A suitable laser provides a wavelength that is absorbed, preferably more strongly absorbed by the film. Such a laser is CO₂Includes lasers, laser diodes, tunable single wavelength Ti: sapphire lasers, and diode-pumped NdYAG. Lasers with ultraviolet to infrared wavelength outputs can be used according to the present invention. In addition to lasers, the adhesive layer can also be activated using electrically heated radiation beaters or heated probes, concentrated or masked non-laser light sources such as flash bulbs, xenon lamps, etc. It is possible. FIG. 8a-8d illustrates the continuous steps of the adhesive transfer method according to one aspect of the invention. As shown in FIG. 8a, the adhesive transfer method of the present invention utilizes a transfer surface that includes a support layer 31 and an active adhesive layer 32. In methods that utilize laser activation of the adhesive layer, the support layer 31 is preferably transparent and is made from, for example, a transparent polymer, glass, or similar material. The active adhesive layer 32 can be an emulsion layer, a coated film, or a separate impregnated web that is secured to the support layer. Examples of materials from which the adhesive layer 32 is manufactured are temperature sensitive adhesives and waxes (eg # HAL-2 from Precision Coatings). 180C), thermal adhesives and sealants (available from Bay Fastening Systems, Brooklyn, NY), UV sensitive or curable optical adhesives (eg NO60-NOA81 from ThorLabs Inc.), and thermal or optical emulsions (eg, NO60-NOA81 from ThorLabs Inc.). For example, silk screen coated emulsion B6 Hi Mesh, Riso Kagaku ) Is included. The support layer 31 provides a physical support for the adhesive surface and can therefore be physically bonded into the active adhesive surface. The activable adhesive layer 32 is characterized by its ability to be stimulated (activated) by electromagnetic rays to become locally adherent to the tissue. To selectively activate the activable adhesive layer 32, one or more chemical components are incorporated into the layer, where the chemical components cause selective absorption of electromagnetic energy. Preferably, such a chemical component is an IR-absorbent dye suitable for use with, for example, a laser diode. As shown in FIG. 8a, the transfer surface 30 is initially located on a cell material sample 33 that can be a microtome fragment or cell smear supported on a support member 34 that can be a microscope slide. For tissue microtomes, paraffin-embedded, formalin-fixed tissue samples can be supplied using conventional methods. As shown in FIG. 8b, the transfer surface 30 is brought into contact with the cell material sample 33. It is noted that the activable adhesive layer 32 preferably has a larger area than the subregion of the cell material sample that is subsequently selected for acquisition. The transfer surface 30 may be secured to the cell material support sample 33 by a clip, adhesive, tape, standard adhesive or similar convenient means. The transfer surface 30 can also include label area 35 (see dotted line in Figure 8b) for writing information, such as patient identification codes or study plans. After the transfer surface 30 is brought into contact with the cell material sample 333, the cell material sample is observed with a standard bottom or high magnification microscope to locate the area of interest "A". This region can range in size from a region smaller than a single cell (less than 10 microns) to a complete portion of several cells, cells or tissues. If the region of interest A is identified, the exact region of the activable adhesive layer 32 that resides just above that region A is a beam of electromagnetic energy 36, as shown in FIG. 8c. , For example activated by a laser beam. The application of the electromagnetic eclipse 36 causes the adhesion of the region of the activable adhesive layer 32 located just above the region "A" to that region "A". Figures 8c and 8d show a single region of interest A, but it is understood that multiple intermittent regions of interest are selected and can be obtained with the proper help and application of electromagnetic energy. Should be. As shown in FIG. 8d, after one or more regions of interest are recognized and that corresponding region of the activable adhesive layer 32 is activated by the bim of electromagnetic energy 36, the transfer surface 30 is cellular. It is detached from the material sample support 34. As shown, the removed transfer surface 30 carries only the exact cell material from the region of interest "A", which is pulled away from the remaining cell material sample. As mentioned above, a single transfer surface can be used to remove many areas of interest from a single cell material sample. The transfer surface 30 carrying the acquired cellular material can be treated with the appropriate reagents to analyze the constituents of the transferred material. This can be achieved by impregnating the transfer surface 30 to which the acquired cellular material is attached with a suitable reagent solution. On the other hand, one or more acquired cell material regions can be removed from the transfer surface 30, or a portion of the transfer surface 30 to which the acquired cell material is attached is punched from the transfer surface 30 and separately. Can be analyzed. In the following examples 4 and 5, the next sample acquisition method was as follows. Formalin-A 5-10 micron fragment of fixed, paraffin-embedded or frozen tissue was prepared on a glass slide according to conventional surgical pathological protocols. Paraffin fragments were deparaffinized with xylene (x2), 95% ethanol (x2), 50% ethanol (x2), distilled water (x2) and air dried. Frozen or paraffin fragments were briefly stained with eosin (1% eosin in 80% ethanol) and air dried. Adjacent hematoxylin and Eosin fragments were used to evaluate tissue fragments for optimal regions of microexcision, namely localization of specific small cell populations of interest, elimination of regions containing significant inflammation, and the like. Microexcisions of selected cell populations were performed under direct light microscopy visualization. A sterilized 30 gauge needle was used as the transfer surface. The electrostatic interaction between the needle and the cell material provides the adhesion required to eliminate the selected cell population. It was determined that a pure cell population of as many as 5 cells could be obtained. In addition, the possibility of acquiring cells arranged as a single cell layer, ie, normal epithelium, epithelial lining of cystic lesions, etc., has been found.<u style="single">Example 4</u>Human prostate cancer has been shown to progress through a field tumor phase called high-grade prostatic intraepithelial neoplasia (PIN) before it progresses to overt invasive cancer. PIN foci have occasionally been found to be associated with prostate cancer, and histologically, cells in PION foci have several features that resemble those of invasive prostate cancer cells. Previous reports have shown that PIN lesions are occasional and aneuploid. However, the exact relationship between PIN and invasive cancer remains unclear. In this example, frozen normal and tumor prostate samples were taken from 100 patients treated by transurethral prostatectomy or radial prostatectomy. Of them, 30 cases containing overt invasive cancer and at least one lesion of identifiable PIN were selected for the study between these cases. Of the cases in the group, 14 cases contained one or more lesions of PIN. Histopathology of tumors varied and included well-differentiated, moderately-differentiated, and poorly-differentiated tumors. PIN lesions were both low and high. Microscopic visualization of selected populations of normal epithelial cells, cells from PIN lesions, and invasive tumor cells from frozen tissue fragments, using the methods discussed above. Conducted below. Specific cells of interest were microexcised and obtained from an unstained 8 μm frozen fragment. In individual cases, normal epithelium, PIN cells, and invasive tumor cells from the same patient were analyzed. The acquired cells were immediately resuspended in 20 ml of a solution containing 10 mM Tris-HCl, pH 8.0, 100 mM ethylenediaminetetraacetic acid (EDTA), 1% Tween 20, 0.1 mg / ml proteinase K, and Incubated overnight at 37 ° C. The mixture was boiled for 5 minutes to inactivate proteinase K, and 0.5-2% of this solution was used for polymerase chain reaction (PCR) analysis. Oligonucleotide Primer D8S136, Chromosome 8p12-21 was positioned using D8S137 and NEFL. Reactions with D8S137 and NEFL were performed in the MJ study thermal cycler as follows: 950 ° C for 2 minutes, followed by 40 cycles in the next cycle: 950 ° C for 30 seconds, 620 ° C for 30 seconds. , 720 ° C for 30 seconds, followed by 720 ° C for the last 2 minutes of incubation. The reaction with D8S136 was performed as follows: 950 ° C for 2 minutes, followed by 40 cycles at 950 ° C: 950 ° C for 30 seconds, 550 ° C for 30 seconds, 720 ° C for 30 Incubate for the last 2 minutes at 720 ° C for a second. PCR was performed with 200 mM dNTP, 0.8 mM primer, 2 μl α [³²P] 12.5 ml of reaction mixture containing dCTP (NEN) and 1 unit of Taq polymerase. The labeled amplified DNA was mixed with an equal volume of formamide-filled dye (95% formamide; 20 mM EDTA; 0.05% bromophenol blue; and 0.05% xylene cyanol). Samples were denatured at 950 ° C for 5 minutes, and 7% acrylamide (49: 1 acrylamide: bis), 5.7M urea, 32% formamide, and 0.089M tris, 0.089M borate, 0.002M. Was loaded into a gel containing EDTA (1 x TBE). Samples were electrophoresed at 95 watts for 2-4 hours. Transfer the gel to 3 mM Whatman paper, and autoradiography to Kodak This was done with X-OMAT film. The criterion for loss of heterozygosity (LOH) was the complete or near complete absence of one allele, as determined by visualization. Patients with LOH showed two alleles in a normal epithelial control and one allele in a tumor or PIN with all similar strengths. Patients with a complete or near complete loss of one allele in the tumor or PIN (ie, a very pale band) were considered positive for LOH with that marker. The method of the invention was used to microcision cells from tissue fragments to study heterozygous loss on chromosome 8p12-21 in patients with both prostate cancer and adjacent lesions of PIN. .. Tissue microexcision was performed on 30 patients with concomitant PIN and invasive prostate cancer. In individual patients, at least one lesion of normal epithelium, invasive prostate cancer and PIN from the same patient was tested. Multiple foci of PIN were tested in 14 patients. In all patients, individual PIN lesions and corresponding invasive tumors were selectively microexcised from adjacent substrates, normal epithelium and inflammatory cells. Acquired a substantially pure cell population of interest. LOH on chromosome 8p12-21 was present in at least one PIN lesion in 26 (89.6%) of the 29 informative patients. Eleven of those patients exhibited different allelic loss patterns between PIN lesions, such as the loss of the opposite allele. In total, 8p12-21 LOH was found in 63.6% (35/55) of the PION lesions studied. Allelic loss of chromosome 8p12-21 was found in 28 (96.5%) of invasive tumors out of 29 patients. Compared to the success associated with the adhesive transfer technique of the present invention, the use of the exfoliation cultivating technique produced less than 15% LOH. This suggests that the sensitivity of the adhesive transfer of the present invention was even higher than that of the conventional technique.<u style="single">Example 5</u>Early-stage on-site breast cancer has occasionally been observed to occur in association with widespread epithelial hyperplasia and invasive cancers. Pathologists have histologically interpreted the common associations of atypical hyperplasia, field cancers and invasive cancers as evidence of relationships between beings. The polymorphic DNA marker used in this example was PYGM located on chromosome 11q13. The reaction was carried out in a thermal cycler as follows: a total of 35 cycles: 94 ° C for 1.5 minutes, 55 ° C for 1 minute, 72 ° C for 1 minute. PCR was performed in a volume of 10 μl, and 1 μl of 10 × PCR buffer (100 mM Tris-HCl, pH 8.3; 500 mM KCl; 15 mM MgCl.₂0.1% w / v gelatin; 2 μl DNA extraction buffer; 50 pM individual primers; 20 nM individual dCTR, dGTP, dTTP, and dATP; 0.2 μl [³²P] dCTP (6000Cl / mM); and 0.1 units of Taq DNA polymerase) were included. The labeled and amplified DNA was mixed with an equal volume of formamide-filled dye (95% formamide; 20 mM EDTA; 0.05% bromophenol blue; and 0.05% xylene cyanol). Samples were denatured at 95 ° C for 5 minutes and loaded into a gel consisting of 6% acrylamide (49: 1 acrylamide: bis). The sample was electrophoresed at 1800 volts for 2-4 hours. Transfer the gel to 3 mM Whatman paper, let it dry, and perform autoradiography in Kodak. This was done with X-OMAT film. The criterion for LOH from microexcised sites and invasive breast samples was the complete absence of alleles. Using the adhesive transfer technique of the present invention, cells were microexcised from normal epithelium, field and invasive cancers from 8 μm thick formalin-fixed, paraffin-depleted fragments from individual biopsies. Allelic deletions on chromosome 11q13 were found in 69% of the human breast cancer cases studied (n = 105). Allelic deletions were observed both at the site of the tumor and at the invasive component. In all cases where the field and invasive cancer were present in the same fragment (26/28), the same allele was lost in the field and invasive cancer. This provides molecular support for the long-supported hypothesis that field breast cancer is a precursor to invasive cancer. To further map the LOH locus on chromosome 11q13, the Genome Center supplied a series of SSCP probes mapped to the corresponding region of chromosome 11. It was determined that the initial LOH region was limited by the proximal marker PYGM and by the distal marker INT-2. Twenty subsets of 105 cases showed either INT-2 or PYGM LOH, but neither. Using those special cases, a series of intervening markers were used to map the smallest overlapping region between INT-2 and PYGM indicating LOH. It was possible to indicate the location of the LOH compartment in a region confined by only one or two YAG or cosmid clones at a location that overlaps the MEN-1 (multiple endocrine neoplasia type I) locus. Sequencing gel analysis was performed on PCR-amplified DNA isolated from microexcised human tissue by the method shown in Figures 8a-8b. The results showed that microexcision of frozen tissue fragments allows for more specific analysis of cell populations within human tumors by conventional techniques. The microexcision technique of the present invention is a pure population of specific cell types. Or it can be used in combination with many different techniques that allow the analysis of enzymes, mRNA and DNA from subpopulations. This simple technique characterizes the distribution of proteases during human tumor invasion, accurately determines protease expression in tumors and / or substrate cell populations as an indicator of tumor aggression, and protease activity at the tumor-substrate interface. It can be used to monitor the effectiveness of anti-protease therapeutic agents in inhibition. In addition, the combination of PCR, RT-PCR, differential display and SSCP with this microexcision technique can identify genetic alterations in specific subpopulations of tumors or substrate cells that are not apparent in heterologous human tumor samples.<u style="single">Example 6</u>Standard 6 μm fragments from formalin or alcohol-fixed, paraffin-embedded storage tissue samples were prepared on uncoated glass slides. Fragments were deparaffinized, stained with hematoxylin and eosin, treated with 3% glycerol in water for 1 minute, and air dried prior to laser capture microexcision (LCM). Fresh tissue, when used, was rapidly frozen at -70 ° C immediately after surgery. 6 μm cold-held fragments were prepared on standard glass on standard histological glass slides. Tissue fragments were fixed in formalin or alcohol and stained with hematoxylin and eosin (Lerner Laboratories, Pittsburgh, PA). Fragments were dehydrated in alcohol and air dried for 5 minutes prior to LCM transfer. For LCM transfer, a 100 μm thick flat film was made by spreading ethylene vinyl acetate melted on a smooth silicone treated or polytetrafluoroethylene surface (Adhesive). Technologies, Hampton, NJ). An optically clear thin film was placed on top of the tissue fragment and the tissue / film sandwich was observed under a reversing microscope (Olympus Model CK2, Tokyo) at 100x magnification (10x objective). A pulsed carbon dioxide laser beam was introduced through a small front mirror coaxial with the condenser optics to illuminate the top surface of the EVA film. Carbon Dioxide Laser (Apollo Company Model 580, Los Angeles, CA or California Laser Company Model LS150, San (Marcos, CA) provided adjustable pulse length and force pulses. The ZnSe lens focused the laser beam on a spot size that could be adjusted on the target specimen. For a transfer spot with a diameter of 150 mm, 25-30 mW was supplied to the film for a 600 msec pulse. For smaller or larger spots, the pulse force was reduced or increased approximately in proportion to the diameter of the laser spot concentrated on the target area. The absorption coefficient of EVA film measured by both the FT-IR spectrometer and direct transmission is about 200 cm at a laser wavelength of 10.6 μm.^-1Met. Since more than 90% of the laser energy was absorbed into the thermoplastic film, there was little direct heating of the tissue specimen. The glass slides provided the targeted tissue with a large cooling radiator that acted to trap the transient moisture of the intensively melted plastic of sufficient thickness. After cooling and recrystallization, the film formed a local surface bond to the targeted tissue that was stronger than the tissue's adhesion to the slide. The film and targeted cells are removed from the tissue specimen, allowing intensive microtransfer of the targeted tissue to the film surface. Immediately remove tissue film and adherent cells for polymerase chain reaction, 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 1% Tween It was resuspended in 40 μl of solution containing 20 and 0.1 mg / ml proteinase K and incubated overnight at 37 ° C. The mixture was then boiled for 10 minutes to inactivate proteinase K. The tube was rotated for a short time (1000 rpm, 1 minute), the film was removed, and 0.5 μl of supernatant was used for PCR. For the most effective transfer recovery, the transfer film is first applied to the tissue fragment as a circular disc with a diameter of about 0.5 cm. After LCM transfer, the disc is placed in wells in a 96-well microtiter plate containing 40 μl of extraction buffer. For polymorphic DNA studies, loci D8S136 and D8S33 located on chromosome 8q, D17S855 located on chromosome 17q21, D11S449 located on chromosome 11q13, D9S171 located on chromosome 9p, exon 2 of the VHL gene Specific primers for Mycobacterium tuberculosis and specific primers for Mycobacterium tuberculosis were used (Research). Genetics, Huntsville, AL). All PCR reactions are for visualization of PCR products³²Incorporation of P-dCTP was included, except for the amplification of M. tuberkilosis visualized by ethidium bromide staining. For reverse transcription-polymerase chain reaction (RT-PCR), total RNA is removed from tissue samples after LCM using a modified method of the published RNA microisolation protocol (Stratagene, La Jolla, CA). Extracted. The volume was adjusted proportionally downwards, and a 10-fold rate of increase (10 ng / ml) in the glycogen carrier was used for all precipitation steps. After initial recovery and resuspension of the RNA pellet, the DNase step was performed at 37 ° with 10 U / ml DNase (Gen Hunter, Nashville, TN) in the presence of 4 units of RNase inhibitor (Perkin Elmer). C was used for 3 hours, followed by RNA re-extraction. RNA sample integrity can be determined using actin-specific primers (Clonetech, Palo Alto, CA), for example by RT-PCR of actin mRNA. Resuspended RNA with 5 μM random hexamer primer (Perkin) Elmer), 250 mM dNTP and 100 units of reverse transcriptase (MMLV, GenHunter, Nashville, TN) were used for reverse transcription. Reverse transcription was achieved by heating the RT mixture (without enzyme) to about 65 ° C for 5 minutes, followed by primer annealing at about 25 ° C for 10 minutes. Reverse transcriptase was then added and then further incubated at 25 ° C for 10 minutes, 37 ° C for 40 minutes, and 94 ° C for 5 minutes. PCR was performed with specific actin or PSA primers and the product was subjected to denatured electrophoresis gel analysis.<u style="single">Example 7</u>A cDNA library was generated using materials isolated by laser capture microexcision. Double-stranded cDNA was prepared from approximately 5 μg of complete cellular RNA based on the RNase H-intervened double-strand replacement method. Reverse transcription first chain synthesis was primed with approximately 50 ng / μl oligo (dT). The reaction was carried out at about 45 ° C for 15 minutes. Second-strand substitutions and EcoRI linker additions were performed as indicated by the manufacturer (Superscript Choice System, Life Technologies Inc., Gaithersburg, MD). After second chain synthesis, the reaction product is electrophoresed and fragments of about 0.3 kb to about 2 kb are agarose (New England). Gels were isolated from 1% low melting point agarose using Biolabs, Beverly, MA). The cDNA pellet was resuspended in 20 μl Tris-EDTA and stored at -20 ° C. 5 μl of the isolated cDNA was amplified by 5 cycles of PCR under standard conditions and a linker-specific primer LINK that also functions to direct UDG cloning. First, the cDNA was denatured at 95 ° C for 3 minutes, followed by 5 degrees by the next cycle: 95 ° C for 15 seconds, 55 ° C for 15 seconds, and 72 ° C for 2 minutes. The final extension was performed at 72 ° C for 5 minutes. Oligonucleotide primers, nucleotides, enzymes, etc. were removed from the reaction mixture by column chromatography (CHROMA SPIN-200, Clontech, Palo Alto, CA). The column effluent was ethanol precipitated and resuspended in 20 μl Tris-EDTA. 6 μl of product, manufacturer's explainer (Life Technologies) It was cloned into the UDG cloning vector pAMP10 according to Inc., Gaithersburg, MD). A complex and relatively low-redundancy library of approximately 200,000 clones was prepared by this means. In contrast to those derived from heterologous tissue or transformed tissue culture cell lines, the cDNA libraries produced under the practice of the invention, whether normal or abnormal, are derived from homogeneous cell types. It has many advantages that cannot be achieved. In addition, the cDNA library allows comparative analysis of mRNA expression during growth, aging, neoplastic transformation, etc. Therefore, the cDNA library of the present invention is also useful for measuring the simultaneous variation in the expression of complex genes or genetic alterations that occur in developing or diseased tissues. To meet the original clinical needs, next-generation molecular analysis methods will be miniaturized and automated. Development of image chips or array systems containing thousands of sequences for automated hybridization is currently underway. Individual chips can be assayed for several possible gene mutations simultaneously, or the expression levels of multiple mRNA species can be measured simultaneously. On the other hand, a series of analytical (SAGE) approaches to gene expression can be used to simultaneously assess the mRNA expression of multiple transcripts. When using PCR amplification and using such novel automation techniques, molecular diagnostic tests can consist of panel tests rather than individual tests that are currently standard in clinical practice. However, even the most sophisticated genetic test methods input DNA, If the RNA or protein is not derived from a pure population of cells exhibiting a characteristic disease form, it will be of limited value. The use of the cDNA library of the present invention overcomes this problem of tissue sample homogeneity. Thus, specific genetic fingerprints can be established for individual individual lesions with such fingerprints that are very useful in diagnosis, prognosis and as a guide to treatment. The present invention presents routine diagnosis of human tumors, such as microremoval of premalignant lesions of all types of cancer, genetic analysis of infectious diseases, gene therapy, tissue transformation, and gene localization of transgenic animals. Applies to analysis. Further applications of this technique include genotypes, cell products, or rare populations of ectoparasites, such as monospheres parasitized by drug-resistant organisms, Hodgkins disease Reed-Sternberg cells, Caposisarcoma cells, stem cells and vascular cells. Includes analysis of. In addition, genetic analysis or identification of microscopically visualized cells of microbial exoparasitism in tissues, lymph nodes or inflamed areas can also be achieved with high accuracy. Although the present invention has been described by specific means, materials and embodiments, those skilled in the art can readily ascertain the essential features of the invention and various improvements, modifications and modifications can be made to the present invention. It can be done to adapt various uses and features within the scope of the invention.