Target activated microtransfer
Inventors
Emmert-Buck, Michael R. • Tangrea, Michael Anthony • Bonner, Robert F. • Chuaqui, Rodrigo • Pohida, Thomas J.
Assignees
HEALTH AND HUMAN SERVICES GOVERNMENT OF United States, AS REPRESENTED BY SERCRETARY OF • US Department of Health and Human Services
Publication Number
US-7709047-B2
Publication Date
2010-05-04
Expiration Date
2023-07-23
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Abstract
A method of removing a target from a biological sample which involves placing a transfer surface in contact with the biological sample, and then focally altering the transfer surface to allow selective separation of the target from the biological sample. In disclosed embodiments, the target is a cell or cellular component of a tissue section and the transfer surface is a film that can be focally altered to adhere the target to the transfer surface. Subsequent separation of the film from the tissue section selectively removes the adhered target from the tissue section. The transfer surface is activated from within the target to adhere the target to the transfer surface, for example by heating the target to adhere it to a thermoplastic transfer surface. Such in situ activation can be achieved by exposing the biological sample to an immunoreagent that specifically binds to the target (or a component of the target). The immunoreagent can alter the transfer surface directly (for example with a heat generating enzyme carried by the immunoreagent), or indirectly (for example by changing a characteristic of the target). In some embodiments, the immunoreagent deposits a precipitate in the target that increases its light absorption relative to surrounding tissue, such that the biological specimen can be exposed to light to selectively heat the target. Alternatively, the immunoreagent is an immunofluorescent agent that carries a fluorophore that absorbs light and emits heat.
Core Innovation
The invention provides methods, systems, and devices for removing a target from a biological sample by using an in situ activating event originating from within the biological sample to focally alter an adjacent transfer surface. This alteration selectively adheres the target to the transfer surface or increases the permeability of the transfer surface to the target, enabling selective removal of the target from the sample. The target may include cells or cellular components in tissue sections, and the transfer surface is often a thermoplastic film that can be focally melted or altered to adhere to the target.
The problem being addressed is the limitation of existing microdissection techniques, like laser capture microdissection (LCM), which rely on external energy sources directed at the transfer surface to selectively adhere targets. These methods require manual, microscope-based selection of individual target cells and are time-consuming, labor-intensive, and not readily automated for high-throughput analysis. Prior techniques may not effectively handle scattered cells where histological identification is challenging. The invention overcomes these issues by employing reagents that localize specifically to the targets and induce alterations in the transfer surface from within the sample, thereby enabling self-identification and efficient, operator-independent target removal.
The disclosed method involves contacting the biological sample with a reagent that specifically binds to the target and produces a change—generally heating the target—to alter the adjacent transfer surface, such as by melting a thermoplastic layer to adhere the target to it. The reagent may include targeting moieties (e.g., antibodies or nucleic acid probes) and activating moieties (e.g., chromophores, enzymes) that convert an external trigger like light into a local activating event, resulting in focal heating or chemical changes that facilitate selective microtransfer. The method allows multiple targets sharing the characteristic for reagent binding to be removed simultaneously without individual microscopic targeting, enabling higher throughput and improved specificity compared to traditional methods.
Claims Coverage
The patent includes 24 independent claims that cover methods of removing targets from biological samples through reagent-induced alteration of transfer surfaces, and devices and systems employing these methods. The main inventive features address target-activated alteration and adhesion mechanisms, types of reagents and transfer surfaces, and irradiation methods for activation.
Selective heating-induced adhesion to transfer surface
The method comprises contacting a biological sample with a reagent that selectively acts on the target, placing a transfer surface adjacent the sample, heating the target to produce a change in the transfer surface to adhere the target, and selectively removing the target by removing the transfer surface with the adhered target or by moving the target through the transfer surface due to increased permeability.
Use of thermoplastic and transfer layers
The transfer surface includes thermoplastic surfaces or films, with options for introducing thermoplastic material layers between the transfer surface and biological sample to facilitate focal adhesion by melting upon target heating.
Activation triggered by electromagnetic radiation
The reagent is responsive to electromagnetic radiation such as light (including flash lamps or lasers) which excites agents (e.g., chromophores or dyes) within the reagent to selectively heat the target and transfer surface, effecting localized adhesion or permeability change.
Reagents including targeting and activating moieties
Reagents comprise targeting moieties that selectively bind the target and activating moieties that produce heat or chemical changes upon external triggering, enabling in situ activation and selective adhesion.
Biological sample types and preparations
Applicable to a variety of biological samples including tissue sections, biopsy materials, cell cultures, and cytology preparations, wherein the transfer substrate can be placed in direct contact, optionally with an intermediate liquid or polymer layer for enhancing adhesion or permeability modifications.
Methods encompassing target identification without microscopy
The method removes the need for microscopic visualization or manual selection of targets because reagent binding and activation provide target self-identification and selective adherence, allowing non-operator dependent, high-throughput microdissection.
Devices for performing target activated transfer
Devices include mounting surfaces and light sources arranged to irradiate samples to activate reagents and transfer surfaces for selective adhesion. Light sources can provide broad area illumination or scanning beams, coordinated with movable mounting surfaces to cover entire samples.
The claims collectively cover target-activated methods for selective removal of biological sample components by reagent-induced changes in transfer surfaces, incorporating reagents with targeting and activating moieties, thermoplastic and permeability-altering transfer substrates, and activation by electromagnetic radiation. The invention also encompasses devices for implementing these methods in an automated or high-throughput fashion.
Stated Advantages
Allows self-identification and selective adherence of target cells, reducing the need for operator manual targeting and visualization.
Enables high-throughput, automated microdissection of multiple targets simultaneously, significantly increasing efficiency over traditional laser capture microdissection (LCM) methods.
Reduces the number of cells needed for molecular profiling, decreasing amplification bias and improving molecular analysis sensitivity.
Facilitates collection of specific cell populations even when histological identification is challenging or impossible.
Supports precise focal alteration of transfer surfaces from within the biological sample, improving specificity and accuracy of target removal.
Permits operator-independent sample processing, decreasing time and labor costs in molecular diagnostics and research.
Documented Applications
Molecular profiling and analysis of isolated cells from pathological tissue sections, including DNA amplification, RNA analysis by RT-PCR, and protein analysis by electrophoresis.
Diagnosis and research of diseases by isolating abnormal cells, tumor markers, or infectious organisms with high specificity from tissue samples.
High-throughput functional genomics and proteomics at the single-cell level, enabling detection of gene expression changes, somatic mutations, and protein variations in normal and diseased cells.
Screening and identification of diagnostic and prognostic markers in cancer progression and other diseases through selective isolation of specific cell populations.
Automated analysis of multiple tissue specimens for molecular biology and clinical research using reagents and transfer substrates in robotically managed workflows.
Use in gene expression assays, allelotyping, loss of heterozygosity analysis, and protein fingerprinting technologies such as SELDI.
Preparation of samples for downstream applications including sequencing, hybridization assays, and enzyme activity measurements.
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