System and method for pre-analytical substrate processing

Abstract

Some embodiments presented in the present disclosure relate to an automated tissue cutting (ATD) system. The ATD system is a one-stop and potentially low-cost system that can cut a substrate from a pathologist's digital or pen markings on a substrate using non-contact and / or mechanical methods to extract formalin-fixed paraffin-embedded (FFPE) tissue samples: (a) only one or more ROIs are to be preserved; (b) nucleic acid content in non-target regions (RONIs) is removed or decomposed, and all tissue samples are collected from a standard microscope substrate into a specific container.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS Technical Field

[0002] The present disclosure relates generally to the fields of histology and / or pathobiology. More specifically, the present disclosure relates to pre-analytical substrate processing systems and related methods, which in some embodiments can be used to separate and isolate regions of interest (ROIs) and regions of non-interest (RONIs) in substrates (e.g., biological substrates such as histological samples). Background Art

[0003] A variety of methods have been proposed to address the cumbersome nature of scraping procedures for preparing and / or processing histological samples for downstream analysis. For example, vacuum sandblasting techniques have been suggested; however, these can only be deployed in industrial settings and therefore require expensive tools and instruments. In addition, there are currently no vacuum sandblasting techniques that are compatible with substrates mounted on microscope slides. Similarly, particle-based sandblasting methods, such as sandblasting with, for example, silica particles, silica-coated particles, or polymer-coated ferromagnetic beads, require that the particles be separated from the target region (ROI) before performing downstream analysis steps.

[0004] The current tissue cutting process is completely manual, using a razor blade to collect the "S" (i.e., ROI) region directly from the substrate. For example, the conventional workflow for substrate submission includes a completely manual process in which the user manually transfers the pathologist's target region marking on a stained slide to an unstained slide using a standard off-the-shelf marker. The user then scrapes the marked target region off the unstained substrate using a razor blade or similar tool and collects it into a container.

[0005] The manual process described limits operator accuracy by relying entirely on the operator's hand / eye coordination, which can affect the consistency and accuracy of tissue scraping. The process also introduces ergonomic / safety issues, as the constant force applied to the glass surface can cause operator lacerations and ergonomic issues (e.g., carpal tunnel).

[0006] Current implementations of digital pathology have improved many aspects of the histology / pathology workflow. However, there are still some important areas of unmet need. One unmet need is that certain substrate presentations cannot be processed digitally using commercial digital pathology systems. Many cases still require manual glass workflow processing. Therefore, a method is needed to transform this process to achieve a fully digital workflow. Summary of the invention

[0007] Some embodiments presented in the present disclosure relate to an automated tissue cutting (ATD) system. The ATD system is a one-stop and potentially low-cost system that can extract formalin-fixed paraffin-embedded (FFPE) tissue samples from a pathologist's digital or pen markings on a substrate using non-contact and / or mechanical methods to: (a) only one or more ROIs as regions to be preserved; (b) remove or decompose nucleic acid content in non-target regions (RONIs) and collect all tissue samples from a standard microscope substrate into a specific container.

[0008] According to certain aspects of the present disclosure, ATD is combined with a digital pathology process to scrape an ROI on a substrate. Also disclosed are systems and methods for collecting desired "S" (i.e., ROI) regions or removing unwanted "X" regions in a feasible manner. The described systems and methods provide a low-cost, flexible system to digitize slide images and simplify downstream scraping processes.

[0009] In some embodiments, the system is capable of performing the following operations: (a) capturing stained and unstained slide images at appropriate magnification, (b) digitizing pen marks into digital marks, (c) executing an object-based algorithm or other algorithm to match the mark coordinates on the substrate to the associated substrate, (d) extracting the marked sample ROI into a container, (e) removing the sample in the RONI or decomposing the nucleic acid in the RONI and collecting all the sample (e.g., tissue) into a container, and (f) lysing the sample and outputting the lysate.

[0010] The technology may be useful because current manual processes may have certain disadvantages or limitations, such as: (a) poor quality; (b) operator limitations; (c) work hazards; (d) ergonomics and / or (e) safety. For example, manual cutting methods are completely dependent on the hand / eye coordination of the individual operator, which may affect the consistency and accuracy of the results. They may also require a dedicated training program to achieve consistent accuracy. Manual cutting with a constant force applied to the glass surface can also cause ergonomic issues for the operator over time; such as wrist injuries, such as carpal tunnel syndrome. Therefore, in many laboratories, operators must also limit the time spent performing manual cutting to help prevent injuries. In terms of safety, there is also the risk of razor blade injury due to blade breakage, for example, the blade may cause lacerations. However, current laboratory processes often use a completely manual method, for example, manually marking the pathologist's target area using a standard off-the-shelf marker, where the end user can scrape the target area on the substrate using a razor blade or similar tool (i.e., a scalpel).

[0011] Automated systems can be used for sample cutting; however, such systems can be expensive and / or low-throughput. MILLISECT TM The system (Roche GmbH) is a milling machine that can automatically collect target areas on a substrate, thus avoiding the above-mentioned problems of manual collection. However, the system is expensive, slow to operate, and may require many systems and operators for high-volume laboratories. Therefore, this system may not be suitable for high-throughput sample analysis. Laser capture cutting systems are also available from several manufacturers (e.g., Leica LMD6-7 system). In these systems, a UV laser is used to cut (e.g., delineate or scribe the boundaries) of the ROI, and an IR laser is used to expand an adhesive cap to remove the ROI from the substrate (e.g., to improve the release of the sample from the substrate); the system also requires a custom substrate that can be collected for subsequent analysis.

[0012] The present disclosure addresses several aspects of sample cutting that may cause difficulties, and embodiments may include varying levels of system complexity. The present systems and methods provide methods for isolating regions of interest (ROIs) on substrates that can provide higher throughput, thereby providing faster sample cutting, while avoiding manual separation and using standard slide equipment.

[0013] The present disclosure includes, for example, a system for processing a sample fixed on a substrate, the system comprising: (a) a support unit for fixing the substrate; (b) a camera placed in close proximity to the support unit; (c) a processing element configured to remove a portion of the sample fixed on the substrate; and (d) a computing device communicatively connected to the support unit.

[0014] According to various aspects, the camera and processing element may include: (a) an image capture engine configured to use the camera to acquire a first image of a first substrate having a first fixed sample and a second image of the second substrate having a second fixed sample, (b) a digital marking engine configured to allow a user to generate a marked image containing the first image and a digital outline of a portion of the first fixed sample, (c) an image overlay engine configured to map the marked image onto the second image so that the image outlines of the first fixed sample and the second fixed sample are rotated and aligned, and (d) a sample removal engine configured to control the positioning of the support unit and the processing element so that only a portion of the second fixed sample within the digital outline of the first fixed sample is removed.

[0015] Thus, some system embodiments herein include a device for digitally marking a mark that delimits the interface between one or more ROIs on a sample (also referred to herein as an "S" sample) and other samples on a slide (referred to herein as "X" samples). Such an interface includes, for example, an algorithm that allows for the creation of virtual marks on a sample by virtually tracking manual marks previously placed on the same sample or on parallel samples that have been manually aligned with the sample, or automatically aligning previously manually marked parallel samples with a target sample, and virtually tracking marks from one sample to another. Such a device also includes the mechanical components required to perform these actions.

[0016] Some system embodiments herein include means for separating one or more ROIs from any X sample on a slide by selectively removing or ablating the X sample without affecting the ROI via a mechanical process. Such means include, for example, a laser, such as a pulsed laser, a water jet, or a source of radio frequency, milling, electric current, ultrasound, micro-sandblasting, micro-bead blasting, particle blasting, sandblasting, ablation, or thermal energy capable of lysing or ablating animal cells. Such means also include the mechanical components required to employ these methods and algorithms, which can specifically direct mechanisms such as lasers or water jets to the X sample while avoiding the ROI.

[0017] Some system embodiments herein include means for isolating one or more ROIs from an X sample by selectively chemically decomposing the X sample or cells or macromolecules therein. Such means include, for example, bleach, strong acids, strong bases, or one or more enzymes that are selectively directed to the X sample rather than the ROIs on the slide, as well as the mechanical components required to employ such methods and algorithms that can specifically direct chemicals to the X sample while avoiding the ROIs.

[0018] In some system embodiments, the system also includes a means for directly collecting one or more ROIs on the substrate. For example, directly can not include marking, because all samples are collected. Once X is killed or removed, a direct method can be used, because all remaining samples (for example, only S area) can be collected. Exemplary means include, for example, particle sandblasting, blades, such as razor blades, scalpels, scrapers, spoons, punches, vacuum sources that allow vacuum to remove samples, or provide competitive surfaces or mediums or solutions (for example, particle micro-sandblasting) for ROI samples. The charged surface or medium or the use of a viscous medium extracts ROI from a slide. Such means also include other mechanical elements that automatically control the use of the above-mentioned elements as required.

[0019] In some embodiments, the system includes means for placing the ROI sample into a container. Exemplary means include, for example, mechanical components that can automatically control the following process: taking the ROI sample removed from the slide and placing it into or onto a container such as a well structure, tube, or vial.

[0020] Multiple aspects of the present disclosure describe a method for processing a sample fixed on a substrate, the method comprising: (a) obtaining a first image of a first substrate having a first fixed sample; (b) obtaining a second image of a second substrate having a second fixed sample; (c) generating a marked image comprising a digital outline of the first image and a portion of the first fixed sample; (d) superimposing the marked image on the second image to align the image outlines of the first fixed sample and the second fixed sample; and (e) using a processing element to remove only a portion of the second fixed sample within the digital outline of the first fixed sample.

[0021] Other aspects of the present disclosure describe a system for processing a sample fixed to a substrate, the system comprising: (a) a support unit for fixing the substrate; (b) a camera placed in close proximity to the support unit; (c) a processing element configured to provide a nucleic acid denaturing agent to denature nucleic acids on a portion of the sample fixed to the substrate; and (d) a computing device communicatively connected to the support unit.

[0022] According to multiple aspects, the camera and the processing element may include: (a) an image capture engine configured to use the camera to obtain a first image of a first substrate having a first fixed sample and a second image of a second substrate having a second fixed sample, (b) a digital marking engine configured to allow a user to generate a marked image containing the first image and a digital outline of a portion of the first fixed sample, (c) an image overlay engine configured to overlay the marked image on the second image to align the image outlines of the first fixed sample and the second fixed sample, and (d) a nucleic acid denaturation engine configured to control the positioning of the support unit and the processing element to denature the nucleic acid only in the X or (RONI) portion of the second fixed sample within the digital outline of the first fixed sample, the nucleic acid denaturation engine comprising a chemical analyzer for performing chemical analysis, a mass spectrometer and / or a cell analyzer for performing cell analysis.

[0023] Another aspect describes a method for processing a sample fixed on a substrate, the method comprising: (a) obtaining a first image of a first substrate having a first fixed sample; (b) obtaining a second image of a second substrate having a second fixed sample; (c) generating a labeled image comprising the first image and a digital outline of a portion of the first fixed sample; (d) superimposing the labeled image on the second image to align the image outlines of the first fixed sample and the second fixed sample; and (e) using a processing element to denature nucleic acids in only a portion of the second fixed sample that is within the digital outline of the first fixed sample.

[0024] In another aspect, systems, methods and devices for processing histological samples, such as FFPE slides or fixed tissues are disclosed. These systems, methods and devices overcome the limitations of existing particle microinjectors (PMB) and computer numerical control (CNC) milling technologies, greatly improving ease of use and the sensitivity and / or specificity of analytical workflows. In particular, the present disclosure relates to variations of PMB and CNC milling technologies that allow for efficient removal of non-target regions (RONIs) or collection of target regions (ROIs) from histological samples while minimizing the loss of analytes (e.g., nucleic acids, proteins, and other macromolecules) in the ROI. The methods described are simple and can be seamlessly integrated with downstream analytical procedures.

[0025] According to the present disclosure, a combined sandblasting technique is used to process biological samples mounted on a substrate such as a glass slide. A combination of sandblasting material (i.e., particles) and compressed air are forced into the system and directed to the patient's tissue ROI. The PMB is configured to contain the sandblasted patient tissue within the walls of the PMB. Next, a vacuum is used to remove the sandblasted tissue from the area and accumulate it in a container. The container can be divided using a filter to establish a vacuum but prevent part of it from reaching the vacuum pump. The method can be used for sandblasting of a waste area ("X") or a target area ("S"), or for sandblasting of all target contents. The sandblasting media can be made of different materials, including, for example, aluminum oxide; silicon dioxide; metal-based particles; magnetic or ferromagnetic particles; salts, ionic particles, and lyophilized reagent particles.

[0026] In contrast to existing PMB techniques that require separation of particles from an intended final target (eg, prior to downstream processing), the systems and methods of the present disclosure do not require separation of particles from a processed sample, as the particles themselves are used for downstream processing.

[0027] The method of the present invention also has advantages over existing methods because it allows for the integration of multiple processing steps into one step, which helps reduce cross-contamination and can also collect analytes, such as nucleic acids, from tissue samples in a highly simplified, efficient and inexpensive manner. By reducing operator considerations, the currently disclosed systems and methods help reduce the variability and / or increase the reproducibility of histological tests (e.g., immunohistochemistry, nuclease protection tests, etc.).

[0028] Another advantage of the present technology is that the systems and / or methods disclosed herein can be modularly applied to existing analytical workflows, such as downstream analytical procedures, such as microscopy, hybridization assays, sequencing, chromatography, spectrometry, ELISA, etc. This means that the currently disclosed systems and / or methods can be seamlessly integrated in various pre-processing and post-processing steps. If desired, the systems and methods can also be applied as scaffolds to various downstream analytical steps.

[0029] The methods of the present invention can be easily modified or adapted to utilize a variety of ablative particles, for example, particles of different sizes, shapes, or materials can be selected based on the preparation and / or composition of the target tissue. For example, depending on the target of interest, alternative material types can be used, such as hydrophobic, polar, non-polar, particles with ion exchange capacity, affinity-specific particles, dielectric particles, lyophilized particles, etc. Moreover, because the reagents and systems used herein are relatively inexpensive, the present technology can be easily integrated into the analytical workflow to significantly improve performance without significantly increasing the cost of the assay.

[0030] Accordingly, this disclosure relates in part to the following non-limiting embodiments:

[0031] In some embodiments, the present disclosure relates to a method of processing a biological sample for a biological assay, the method comprising: (a) contacting the biological sample with a contact medium comprising particulate matter and compressed air under conditions sufficient to achieve at least partial transfer of components in the biological sample to the contact medium; and (b) removing the contact medium from the biological sample. Preferably, the biological sample is processed for analysis of one or more analytes of diagnostic interest.

[0032] The present disclosure relates to a method for treating a biological sample for bioassay according to the foregoing or following embodiments, for example, for analyzing one or more diagnostic analytes, wherein the biological sample comprises a punch biopsy sample, a needle biopsy sample, fresh tissue, tissue culture, a frozen tissue specimen, a tissue treated with neutral formalin, an organ, a cell organelle, formalin-fixed paraffin-embedded (FFPE) tissue, wax-fixed embedded tissue, ethanol-fixed paraffin-embedded (EFPE) tissue, hematoxylin and eosin (H&E) stained tissue, or glutaraldehyde-fixed tissue. Preferably, the biological sample comprises FFPE or EFPE tissue. Preferably, the biological sample comprises cells from tumor tissue, degenerative tissue, inflamed tissue (e.g., tissue from a patient suffering from an inflammatory disease such as rheumatoid arthritis, ulcerative colitis, Crohn's disease, etc.).

[0033] The present disclosure relates to a sample milling device including a first and a second component. The first component has openings at both ends. The second component is fixed to one end of the first component and has a sample collection port, which is facing away from the position where the second component is fixed to the first component. The sample collection port has one or more sample scraping elements protruding along the periphery of the sample collection port. A vacuum channel extends through the first and second components to connect the sample collection port to a vacuum connection port on the other end of the first component.

[0034] The present disclosure relates to a method for processing a biological sample for biological assay (e.g., for analyzing one or more analytes of diagnostic interest) according to the preceding or following embodiments, comprising contacting a target region (ROI), a non-target region (RONI), or all regions in the biological sample with a contact medium; preferably, contacting the target region (ROI) with the contact medium.

[0035] The present disclosure relates to a method for processing a biological sample for biological assay (e.g., for analyzing one or more diagnostic analytes) according to the preceding or following embodiments, wherein the biological sample comprises at least one diagnostic analyte selected from genomic DNA (gDNA), methylated DNA, specific methylated DNA, messenger RNA (mRNA), fragmented DNA, fragmented RNA, fragmented mRNA, mitochondrial DNA (mtDNA), chloroplast DNA (ctDNA), viral RNA or viral DNA, microRNA, ribosomal RNA, in situ PCR product, polyA mRNA, RNA / DNA hybrid, lipid, carbohydrate, protein, glycoprotein, lipoprotein, phosphoprotein, specific phosphorylated or acetylated variant of protein, or viral coat protein; preferably a nucleic acid selected from mRNA, gDNA, viral DNA or viral RNA.

[0036] The present disclosure relates to a method of processing a biological sample for a biological assay (e.g., for analyzing one or more analytes of diagnostic interest) according to the preceding or following embodiments, wherein the particulate matter in the blasting media comprises aluminum oxide; silicon dioxide; metal-based particles; magnetic or ferromagnetic particles, or a combination thereof.

[0037] The present disclosure relates to a method for processing a biological sample for biological assay (e.g., for analyzing one or more analytes of diagnostic interest) according to the preceding or following embodiments, wherein the particulate matter is capable of binding to an analyte or non-analyte in the biological sample; preferably, the particulate matter is capable of binding to the analyte through an interaction selected from ionic interactions, polar-nonpolar interactions, hydrophobic interactions, van der Waals interactions, chemical coupling, dielectric or zwitterionic interactions, or a combination thereof.

[0038] The present disclosure relates to a method of processing a biological sample for a biological assay (e.g., for analyzing one or more analytes of diagnostic interest) according to the preceding or following embodiments, wherein the contact medium comprises a compressed gas selected from pressurized helium, argon, xenon, nitrogen, carbon dioxide, or a combination thereof.

[0039] The present disclosure relates to a method for processing a biological sample for biological assay (e.g., for analyzing one or more analytes of diagnostic interest) according to the preceding or following embodiments, wherein the biological sample is mounted on a substrate, such as a substrate selected from glass, silicon, poly-L-lysine coated material, nitrocellulose, polystyrene, cyclic olefin copolymer (COC), cyclic olefin polymer (COP), polypropylene, polyethylene, paper and / or polycarbonate.

[0040] The present disclosure relates to a method of processing a biological sample for a biological assay, e.g., for analyzing one or more analytes of diagnostic interest, according to the preceding or following embodiments, wherein the biological sample comprises a nucleic acid analyte and the contact medium comprises a particulate material comprising silica.

[0041] The present disclosure relates to a method of processing a biological sample for biological assays, eg, for analysis of one or more analytes of diagnostic interest, according to the preceding or following embodiments, and also comprising microdissection, eg, laser microdissection.

[0042] The present disclosure relates to a method of processing a biological sample for a biological assay according to the preceding or following embodiments, e.g., for analysis of one or more analytes of diagnostic interest, wherein a contact medium is removed from the biological sample by vacuum treatment, a pressure differential or gradient, gravity, a transport medium (e.g., a liquid, an aerosol, or a gas), or a transfer medium selected from a magnetic field or an electric field.

[0043] The present disclosure relates to a method for processing a biological sample for biological assay according to the preceding or following embodiments, for example, for analyzing one or more analytes of diagnostic interest, the method comprising: (a) selectively contacting a non-target region (RONI) in the biological sample with a contact medium, wherein the selective contacting preferably includes leaving a target region (ROI) in the biological sample uncontacted; (b) selectively contacting a target region (ROI) in the biological sample with a contact medium, wherein the selective contacting preferably includes leaving the non-target region (RONI) in the biological sample uncontacted; or (c) contacting both the ROI and the RONI in the biological sample with a contact medium.

[0044] The present disclosure relates to a method for processing a biological sample for biological assays according to the preceding or following embodiments, for example, for analyzing one or more analytes of diagnostic purpose, comprising collecting particulate matter in a contact medium; (c) optionally preparing the particulate matter for analysis; and (d) further optionally analyzing the particulate matter.

[0045] The present disclosure relates to a method for processing a biological sample for a biological assay according to the preceding or following embodiments, e.g., for analyzing one or more analytes of diagnostic purpose, wherein preparing the particulate matter for analysis comprises treating the particulate matter with a buffer (e.g., a lysis buffer) and washing the particulate matter to remove non-analytes. Preferably, in this embodiment, the analysis of particulate matter includes polymerase chain reaction (PCR), quantitative PCR (qPCR), reverse transcriptase PCR (RT-PCR), nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP), rolling circle amplification (RCA), immunoassay, immunoPCR (iPCR), enzyme activity assay, staining, imaging, whole genome amplification (WGA), in situ PCR, in situ WGA, colony formation, sequencing, single molecule sequencing, nanopore analysis, nanopore sequencing, single molecule imaging, DNA ball formation, electrophoresis, microelectromechanical system (MEMS) electrophoresis, mass spectrometry, chromatography (e.g., HPLC), proximity ligation assay, electrochemical detection, plasmon resonance (SPR), hybridization assay (e.g., in situ hybridization assay, such as fluorescence in situ hybridization (FISH)) FRET, cell sorting (e.g., FACS), electrochemiluminescent ELISA and chemiluminescent ELISA.

[0046] The present disclosure relates to a method for treating a biological sample for biological assay according to the preceding or following embodiments, for example, for analyzing one or more analytes of diagnostic interest, wherein the method further comprises mixing a contact medium with an enrichment medium. Preferably, in this embodiment, the enrichment medium comprises a substance that specifically binds to a target analyte in the biological sample, such as an antibody that specifically binds to a target protein antigen or a nucleic acid that specifically hybridizes to a target nucleic acid.

[0047] The present disclosure relates to a method for processing a biological sample for biological assays according to the preceding or following embodiments, e.g., for analyzing one or more analytes of diagnostic interest, wherein the biological sample comprises a two-dimensional tissue (e.g., a tissue section or slice) or a three-dimensional tissue (e.g., a tissue block) comprising a region of interest (ROI) and / or a region of non-target (RONI); preferably comprising well-defined spatial locations of both the ROI and the RONI.

[0048] In some embodiments, the present disclosure relates to a method for determining an analyte in a biological sample, the method comprising treating the biological sample by: (a) contacting the biological sample with a contact medium comprising particulate matter and compressed gas under conditions sufficient to achieve at least partial transfer of a component in the biological sample to the contact medium; (b) removing the contact medium from the biological sample to obtain a treated biological sample; and analyzing the analyte in the treated biological sample or the removed contact medium.

[0049] In some embodiments, the present disclosure relates to a bioanalysis system comprising: (a) a first component that contacts a biological sample or a region thereof with a contact medium comprising particulate matter and compressed gas under conditions sufficient to achieve at least partial transfer of components in the biological sample to the contact medium; (b) a second component that removes the contact medium from the biological sample; and (c) optionally a third component that analyzes components in the contact medium or the treated biological sample or both the contact medium and the treated biological sample.

[0050] The present disclosure relates to a bioanalysis system of the preceding or following embodiments, wherein the first component comprises a pressurized particle microinjector (PMB) containing a contact medium.

[0051] The present disclosure relates to a bioanalysis system of the preceding or following embodiments, wherein the second component comprises a vacuum, a pressure differential or gradient, a medium for transporting particulate matter (eg, liquid or aerosol), or a transmission medium selected from a magnetic field or an electric field.

[0052] The present disclosure relates to a bioanalysis system of the preceding or following embodiments, wherein optionally the third component comprises an instrument selected from the group consisting of polymerase chain reaction (PCR), quantitative PCR (qPCR), reverse transcriptase PCR (RT-PCR), nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP), rolling circle amplification (RCA), immunoassay, immuno-PCR (iPCR), enzyme activity assay, staining, imaging, whole genome amplification (WGA), in situ PCR, in situ WGA, colony formation, sequencing, single molecule sequencing, nanopore analysis, nanopore sequencing, single molecule imaging, DNA ball formation, electrophoresis, microelectromechanical system (MEMS) electrophoresis, mass spectrometry, chromatography (e.g., HPLC), proximity ligation assay, electrochemical detection, plasmon resonance (SPR), hybridization assay (e.g., in situ hybridization assay, such as fluorescence in situ hybridization (FISH)) FRET, cell sorting (e.g., FACS), electrochemiluminescent ELISA, and chemiluminescent ELISA.

[0053] Some embodiments presented in the present disclosure relate to an automated tissue cutting (ATD) system. The ATD system is a one-stop and potentially low-cost system that can extract formalin-fixed paraffin-embedded (FFPE) tissue samples from a pathologist's digital or pen markings on a substrate using non-contact and / or mechanical methods to: (a) only one or more ROIs as regions to be preserved; (b) remove or decompose DNA in non-target regions (RONIs), and collect all tissue samples from a standard microscope substrate into a specific container. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 Schematic diagrams showing various doctor blade methods used according to various embodiments.

[0055] Figure 2 A flow chart is provided showing an exemplary process for digitally processing substrate presentation according to various embodiments.

[0056] Figure 3 A flow chart is provided showing an improved method for collecting "S" regions or removing "X" regions according to various embodiments.

[0057] Figure 4 Exemplary methods for sample cutting according to various embodiments are shown.

[0058] Figure 5 Exemplary transmission and reflected radiation of incident radiation on the mask according to various embodiments are shown. This is because the radiation is partially blocked by the mask and the radiation can only reach certain areas (eg, only a certain percentage of energy can be transmitted as a function of wavelength).

[0059] Figure 6 An exemplary slide with marked unwanted "X" regions and regions of interest (ROI) according to various embodiments is shown. For example, the "X" regions and ROIs can be determined by the process described below.

[0060] Figure 7 A process of removing unwanted "X" regions from a substrate by covering the substrate with a mask according to various embodiments is shown. For example, a mask may be prepared as described below. The mask may be covered on the substrate, and the "X" regions may be removed by various methods as described below.

[0061] Fig. 8A and 8B Representative diagrams of particle microinjectors are shown according to various embodiments. Fig. 8A A representative implementation of a PMB of the present disclosure for processing a patient sample is shown; Figure 8B A representative implementation of a PMB of the present disclosure for processing a patient sample is shown.

[0062] Fig. 9 An exemplary system architecture of a computer system for implementing the described systems and methods is shown.

[0063] Fig.10 Exemplary sample processing devices according to various embodiments are shown.

[0064] Fig.11 Exemplary sample processing devices according to various embodiments are shown.

[0065] Fig.12 Exemplary sample processing systems are shown according to various embodiments. DETAILED DESCRIPTION

[0066] definition

[0067] As described in this section, certain terms used herein are defined. Other terms are defined or exemplified elsewhere in this disclosure. Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs.

[0068] In this application, the use of "or" means "and / or" unless stated otherwise. In the context of multiple dependent claims, the use of "or" refers only to one or more prior independent or dependent claims among the alternative claims.

[0069] The word "about" means a range of plus or minus 10% of the value, for example, "about 5" means 4.5 to 5.5, "about 100" means 90 to 100, etc., unless the context of the present disclosure indicates otherwise or is inconsistent with such an interpretation. For example, in a numerical list such as "about 49, about 50, about 55", "about 50" refers to a range extending to less than half of the interval between the previous value and the subsequent value, for example, greater than 49.5 to less than 52.5. In addition, the phrases "less than about" a numerical value or "greater than about" a numerical value should be understood in view of the definition of the term "about" provided herein.

[0070] Where a numerical range is provided in the present disclosure, it is intended that each intermediate value between the upper and lower limits of the range and any other stated or intermediate value in the stated range is included in the present disclosure. For example, if the stated range is 1 μm to 8 μm, it is intended that 2 μm, 3 μm, 4 μm, 5 μm, 6 μm and 7 μm are also disclosed.

[0071] As used herein, the term "plurality" may be 2, 3, 4, 5, 6, 7, 8, 9, 10 or more.

[0072] As used herein, the term "detection" refers to the process of determining a value or a set of values ​​associated with a sample by measuring one or more parameters of the sample, and may further include comparing the test sample to a reference sample. According to the present disclosure, the detection of a tumor includes identifying, determining, measuring and / or quantifying one or more markers.

[0073] As used herein, the term "likelihood" generally refers to probability, relative probability, presence or absence, or degree.

[0074] As used herein, the terms "comprises" (or variations thereof), "contains" (or variations thereof), "has" (or variations thereof), or "includes" (or variations thereof) are not intended to be limiting, but are inclusive or open-ended, and do not exclude other uncited additives, components, integers, elements, or method steps. For example, a process, method, system, composition, kit, or device that includes a list of features is not limited to only those features, but may also include other features not expressly listed or inherent to such process, method, system, composition, kit, or device.

[0075] As used herein, the term "sample" refers to a composition obtained from or derived from a target subject, which includes, for example, cells and / or other molecular entities characterized and / or identified based on physical, biochemical, chemical and / or physiological characteristics. Preferably, the sample is a "biological sample", which refers to a sample derived from a living body such as a cell, tissue, organ, etc. In some embodiments, the source of the tissue sample can be blood or any blood component; body fluid; solid tissue from a fresh, frozen and / or preserved organ or tissue sample or biopsy or aspirate; and cells or plasma from any time during the pregnancy or development of the subject. Samples include, but are not limited to, primary or cultured cells or cell lines, cell supernatants, cell lysates, platelets, serum, plasma, liquids (e.g., lymph, amniotic fluid, milk, whole blood, urine, CSF, saliva, sputum, tears, sweat, mucus, tumor lysates, and cell culture media), homogenized tissues, tumor tissues, and cell extracts. Samples also include biological samples that have been treated, dissolved or enriched for certain components (e.g., proteins or nucleic acids), or biological samples embedded in semisolid or solid matrices for sectioning purposes, such as thin sections of tissue or cells in histological samples. The sample may include environmental components, such as water, soil, mud, air, resin, minerals, etc. Preferably, the biological sample comprises DNA (e.g., gDNA, mtDNA), RNA (e.g., mRNA, tRNA), protein, or a combination thereof obtained from a subject (e.g., a human or other mammalian subject).

[0076] As used herein, the term "cell" is used interchangeably with the term "biological cell". Non-limiting examples of biological cells include eukaryotic cells, plant cells, animal cells (e.g., mammalian cells, reptile cells, avian cells, fish cells, etc.), prokaryotic cells, bacterial cells, fungal cells, protozoan cells, etc., cells dissociated from tissues (e.g., muscle, cartilage, fat, skin, liver, lung, neural tissue, etc.), immune cells (e.g., T cells, B cells, natural killer cells, macrophages, etc.), embryos (e.g., fertilized eggs), oocytes, egg cells, sperm cells, hybridomas, cultured cells, cells from cell lines, cancer cells, infected cells, transfected and / or transformed cells, reporter cells, etc. Mammalian cells can be, for example, from humans, mice, rats, horses, goats, sheep, cattle, primates, etc.

[0077] As used herein, the term "tumor" includes any cell or tissue that may have undergone transformation at the genetic, cellular, or physiological level compared to a normal or wild-type cell. The term generally refers to a neoplastic growth that may be benign (e.g., a tumor that does not form metastases and destroys adjacent normal tissue) or malignant / cancerous (e.g., a tumor that invades surrounding tissue and is often capable of producing metastases, may recur after attempted removal, and is likely to result in death of the host unless appropriately treated). See Steadman's Medical Dictionary, 28 th Ed Williams & Wilkins, Baltimore, MD (2005).

[0078] The term "cancer" refers to abnormal cell growth, particularly cancers and carcinomas that are malignant in nature, sarcomas, adenocarcinomas, lymphomas, leukemias, solid and lymphomas, etc. Examples of different types of cancer include, but are not limited to, lung cancer, pancreatic cancer, breast cancer, stomach cancer, bladder cancer, oral cancer, ovarian cancer, thyroid cancer, prostate cancer, uterine cancer, testicular cancer, neuroblastoma, squamous cell carcinoma of the head, neck, cervix and vagina, multiple myeloma, soft tissue and bone sarcomas, colorectal cancer, liver cancer, kidney cancer (e.g., RCC), pleural cancer, cervical cancer, anal cancer, bile duct cancer, gastrointestinal carcinoid tumors, esophageal cancer, gallbladder cancer, small intestine cancer, central nervous system cancer, skin cancer, choriocarcinoma, osteogenic sarcoma, fibrosarcoma, glioma, melanoma, etc.

[0079] The term "normal" used in the context of "normal cells" refers to cells of an untransformed phenotype or cells that exhibit the morphology of untransformed cells of the tissue type being examined (e.g., PBMCs). In some embodiments, "normal samples" as used herein include non-tumor samples, such as saliva samples, skin samples, hair samples, etc. It should be noted that the methods of the present disclosure can be implemented without the use of normal samples.

[0080] As used herein, the term "abnormal" generally refers to a state of a biological system that deviates from normal (e.g., wild type) to some extent. Abnormal states can occur at the physiological or molecular level. Representative examples include, for example, physiological states (diseases, pathologies) or genetic aberrations (mutations, single nucleotide variants, copy number variants, gene fusions, indels, etc.). The disease state can be cancer or precancer. Abnormal biological states may be related to the degree of abnormality (e.g., a quantitative measurement indicating a distance away from the normal state).

[0081] As used herein, the term "marker" refers to a characteristic that can be objectively measured as an indicator of a normal biological process, a pathogenic process, or a pharmacological response to a therapeutic intervention (e.g., treatment with an anticancer agent). Representative types of markers include, for example, molecular changes in the structure (e.g., sequence) or number of markers, including, for example, gene mutations, gene duplications, amino acid substitutions, additions or deletions, or multiple differences, such as somatic changes in DNA, copy number variations, tandem duplications, translocations, or combinations thereof.

[0082] As used herein, the term "genetic marker" refers to a polynucleotide sequence having a specific position on the genome or corresponding to a specific position in the genome (e.g., a transcript complementary to the sequence of the genomic position). Therefore, the term "genetic marker" can also be used to refer to, for example, cDNA and / or mRNA encoded by a genomic sequence, and the genomic sequence itself. Genetic markers can include two or more alleles or variants. Genetic markers include nucleic acid sequences that encode or do not encode gene products (e.g., proteins). In particular, genetic markers include single nucleotide polymorphisms / variations or copy number variations or combinations thereof. Preferably, compared with a reference sample, genetic markers include somatic variations in DNA, such as sSNV or sCNV, indels, SV or combinations thereof.

[0083] As used herein, the term "protein marker" or "proteomic marker" refers to a sequence of a polypeptide or fragment thereof (e.g., a biologically active fragment of a polypeptide) corresponding to a transcript (e.g., encoded by a transcript), which in turn corresponds to a genomic sequence (e.g., a transcript transcribed from a DNA sequence).

[0084] "Formalin-fixed wax-embedded" or "Formalin-fixed paraffin-embedded" or "FFPE" tissue samples herein are broadly interpreted as referring to samples that have been fixed with formalin or an equivalent substance and embedded in wax (e.g., paraffin or an equivalent substance). The FFPE tissues herein can be from any human, animal, or plant source.

[0085] A "slide" herein may be any type of surface capable of holding FFPE tissue for analysis and may be made of any suitable material.

[0086] "Region of interest" or "ROI" herein refers to a portion of a sample on a substrate that a user may wish to analyze, for example, to assess changes in the sequence, structure, or expression level of a gene. A sample on a substrate may include all ROIs, no ROIs, one ROI, or more than one ROI. ROIs are also referred to herein as "S" samples. RONI samples on a substrate are referred to herein as "X" samples.

[0087] As used herein, the term "particulate" material refers to a material consisting of particles, such as substantially spherical particles or particles of more irregular shape. Typically, the diameter of the particulate material is about 10nm to about 100μm; preferably, about 50 to about 400nm; especially about 100 to about 200nm.

[0088] As used herein, the term "assay" is a test for the amount, presence or absence of a substance.

[0089] As used herein, the term "compressed" gas refers to a gas that has been compressed, for example, to a pressure greater than atmospheric pressure. The "gas" component in such compression is typically an inert gas selected from helium, argon, xenon, nitrogen, carbon dioxide, or mixtures thereof. As is typical in compression systems, the "gas" component may be in liquid, semi-liquid, or gaseous form.

[0090] As used herein, "contacting" refers to introducing a composition comprising an agent (e.g., a contact medium) into a sample comprising a target (e.g., a cellular target) in a test tube, flask, tissue culture, chip, array, plate, microplate, capillary, etc., and incubating at a temperature and for a time sufficient to allow interaction between the target and the agent.

[0091] In the context of in vivo diagnosis or treatment, "contacting" means introducing an active ingredient (eg, a compound or drug) into a subject and bringing the active ingredient into contact with a target tissue (eg, epithelial tissue) of the subject in vivo.

[0092] As used herein, the term "subject" refers to any animal, preferably a mammal, such as a human, a veterinary or farm animal, a livestock or a pet, including animals commonly used in clinical research. In particular, the subject is a human subject, for example, a human patient diagnosed with a disease such as cancer. The subject may have, potentially have or be suspected of having one or more characteristics associated with the disease, one or more symptoms associated with the disease, be asymptomatic or undiagnosed with respect to the disease. In particular, the subject may have cancer, the subject may show symptoms associated with cancer, the subject may not have symptoms associated with cancer, or the subject may not have been diagnosed with cancer.

[0093] As used herein, the term "noise" refers in the broadest sense to any unwanted interference (e.g., a signal not directly related to a real event) that is processed or received as a real event. Noise is the sum of unwanted or interfering energy introduced into a system by both man-made and natural sources. Noise distorts the signal, thereby degrading or reducing the reliability of the information carried by the signal. The term is contrasted with "signal", which is a function that conveys information about the behavior or properties of a phenomenon, such as the probabilistic association between a marker (SNV, CNV, indel, SV) and a disease (e.g., cancer).

[0094] As used herein, in the context of marker levels, the term "estimate" is used in a broad sense. Thus, the term "estimate" can refer to an actual value (e.g., 1 variation per mbp DNA), a range of values, a statistical value (e.g., mean, median, etc.), or other estimation methods (e.g., probabilistically).

[0095] As used herein, the term "substantially" means sufficient for the intended purpose. Thus, the term "substantially" allows for minor, insignificant variations from an absolute or perfect state, dimension, measurement, result, etc., which are expected by one of ordinary skill in the art but do not significantly affect overall performance. When used with respect to a numerical value or a parameter or characteristic that can be expressed as a numerical value, "substantially" means within ten percent.

[0096] As used herein, the term "component" refers to the composition of a system. For example, in a cell system, components may include polypeptides (e.g., small peptides as well as large proteins), nucleic acids (e.g., DNA or RNA), carbohydrates (e.g., monosaccharides as well as macromolecules such as starch), lipids, and other ingredients such as vitamins and cholesterol.

[0097] As used herein, the term "transfer" is used in the broadest sense to refer to the movement of a component of a system from its native environment (e.g., mitochondria in the case of mitochondrial DNA) to a non-native environment (e.g., the surface of a silica particle) by processes such as binding, bonding, adsorption, etc. The term includes processes such as covalent or non-covalent interactions between the component and the non-native system.

[0098] As used herein, the term "covalent" interaction involves sharing electrons between bonded atoms. In contrast, "non-covalent" interactions may include, for example, ionic interactions, electrostatic interactions, hydrogen bonding interactions, physicochemical interactions, van der Waals forces, Lewis acid / Lewis base interactions, or a combination thereof.

[0099] As used herein, the term "analyte" generally refers to a target molecule detected using a method or system disclosed herein. The analyte can be a DNA analyte, an RNA analyte, a nucleic acid analyte, a macromolecule or a small molecule, such as those terms used in the art. In particular, the macromolecule can include, for example, a polynucleotide, a polypeptide, a carbohydrate, a lipid, or a combination of one or more of these. Typically, the molecular weight of the macromolecule is at least about 300 daltons, and can be millions of daltons. A small molecule is an organic compound with a molecular weight of up to about 300 daltons. In some cases, the analyte is a nucleic acid analyte.

[0100] As used herein, "probe" is a substance, such as a molecule, that can recognize or be specifically recognized by a particular target. Types of potential probe / target or target / probe binding partners include receptor / ligand; ligand / antiligand; nucleic acid (polynucleotide) interactions, including DNA / DNA, DNA / RNA, PNA (peptide nucleic acid) / nucleic acid; enzymes, other catalysts or other substances with substrates, small molecules or effector molecules, etc. Examples of probes contemplated by the present invention include, but are not limited to, peptides, enzymes (such as proteases or kinases), enzyme substrates, cofactors, drugs, lectins, sugars, nucleic acids (including oligonucleotides, DNA, RNA, PNA or modified or substituted nucleic acids), oligosaccharides, proteins, enzymes, polyclonal and monoclonal antibodies, single-chain antibodies or fragments thereof. The probe polymer can be linear or circular. The probe can distinguish different targets by differential activity, differential binding or identification by structural markers. The probe of the present invention is preferably a nucleic acid molecule, and DNA is particularly preferred. In some cases, a "probe" can function as a "target" and a "target" can function as a probe, e.g., complementary DNA (cDNA) can be used as a probe that hybridizes to a portion of a target gene sequence; however, the cDNA itself corresponds to the target sequence because it matches the mRNA product of the gene sequence.

[0101] As used herein, the term "analyzing" as well as the phrase "detecting" may refer to the qualitative or quantitative determination of a parameter of interest associated with an analyte, for example, the amount, level, concentration, or activity (absolute and relative) of the analyte.

[0102] As used herein, the term "diagnosis" refers to a method that can determine whether a subject is likely to suffer from a given disease or condition. Technicians typically diagnose based on one or more diagnostic indicators (e.g., markers), the presence, absence, amount, or change in amount of which indicates the presence, severity, or absence of a disease or condition. Other diagnostic indicators may include patient history; physical symptoms, such as unexplained weight loss, fever, fatigue, pain, or skin abnormalities; phenotype; genotype; or environmental or genetic factors. Those skilled in the art will understand that the term "diagnosis" refers to an increased likelihood of a certain process or outcome; that is, a patient who exhibits a given feature (e.g., the presence or level of a diagnostic indicator) is more likely to have a course of disease or outcome than an individual who does not exhibit the feature. The diagnostic methods of the present disclosure can be used independently or in combination with other diagnostic methods to determine whether a patient who exhibits a given feature is more likely to have a course of disease or outcome.

[0103] The term "nucleic acid" generally refers to DNA or RNA, whether it is an amplification product, synthetically produced, an RNA reverse transcription product or naturally occurring. Typically, nucleic acids are single-stranded or double-stranded molecules and are composed of naturally occurring nucleotides. Double-stranded nucleic acid molecules can have 3' or 5' overhangs, so it is not necessary or assumed to be completely double-stranded over its entire length. In addition, the term nucleic acid can be composed of non-naturally occurring nucleotides and / or modifications to naturally occurring nucleotides. Examples are listed herein, but are not limited to: phosphorylation of 5' or 3' nucleotides to allow attachment or prevent exonuclease degradation / polymerase extension, respectively; covalent and near-covalently attached amino, thiol, alkyne or biotinyl modifications; fluorophores and quenchers; phosphorothioate, methylphosphonate, phosphoramidate and phosphate attachment between nucleotides to prevent degradation; methylated and modified bases.

[0104] When used in this article, the term "polypeptide" refers to interchangeable peptides, proteins or polypeptides, which encompass amino acid chains of a given length, wherein amino acid residues are connected by covalent peptide bonds. The term polypeptide also refers to and does not exclude the modification of polypeptides. Modifications include glycosylation, acetylation, acylation, phosphorylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of heme moieties, covalent attachment of nucleotides or nucleotide derivatives, covalent attachment of lipids or lipid derivatives, covalent attachment of phosphatidylinositols, cross-linking, cyclization, disulfide bond formation, demethylation, covalent cross-linking formation, cysteine ​​formation, pyroglutamic acid formation, preparations, γ-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic treatment, phosphorylation, prenylation, racemization, selenization, sulfation, transfer RNA-mediated amino acids add protein (e.g., arginization) and ubiquitination.

[0105] As used herein, the term "isolated" or "extracted" in the context of a molecule refers to a molecule that is substantially free of impurities. A molecule (e.g., DNA or RNA) has been "isolated" or "extracted" when it is purified from other components in a sample. Purification refers to the separation of a target from one or more extraneous components that are also found in a sample. An isolated, extracted or purified component from a mixed specimen or sample is typically purified or enriched by at least 50%, at least 60%, at least 75%, at least 90%, or at least 98%, or even at least 99%, compared to an unpurified or unextracted sample.

[0106] The term "synthetic" refers to molecules that have been chemically synthesized using techniques understood in the art, for example, using phosphoramidite chemistry or synthetic chemistry.

[0107] In the context of nucleic acids, the term "hybridization" or "hybridization" is broadly meant to include duplexes as well as molecules capable of forming duplexes in this manner. In this article, a single-stranded nucleic acid that base pairs at multiple bases is referred to as a "hybridization." Hybridization is typically determined under physiological or biological conditions (e.g., intracellular: pH 7.2, 140 mM potassium ions; extracellular: pH 7.4, 145 mM sodium ions).

[0108] As used herein, the term "analog" includes, but is not limited to, oligonucleotides having synthetically introduced residues or linkers, such as ribonucleic acid residues within a DNA sequence, branched linkers such as glycerol derivatives, or aminoalkyl linkers. "Adducts" include, for example, O6-alkyl-dG and O6-Me-dG. Similarly, in one embodiment, the term "conjugate" refers to a target recognition agent covalently or non-covalently bound to one or more polynucleotides. In another embodiment, the term "conjugate" refers to a linear, branched or dendritic polynucleotide covalently or non-covalently bound to one or more fluorescent dye molecules.

[0109] As used herein, "target" refers to a substance whose presence, activity and / or amount need to be determined and has affinity for a given probe. Targets can be artificial or naturally occurring substances. Moreover, they can be used in an unchanged state or used together with other species as aggregates. The target can be covalently or non-covalently attached to a binding member directly or through a specific binding substance. Examples of targets that can be used in the present invention include, but are not limited to, nucleic acids or polynucleotides (including mRNA, tRNA, rRNA, oligonucleotides, DNA, viral RNA or DNA, EST, cDNA, PCR amplification products derived from RNA or DNA, and mutations, variants or modifications thereof); proteins (including enzymes, such as enzymes responsible for cutting neurotransmitters, proteases, kinases, etc.); substrates of enzymes; peptides; cofactors; lectins; sugars; polysaccharides; cells (may include cell surface antigens); cell membranes; organelles, etc., and other such molecules or other substances that can exist in the form of complexation, covalent cross-linking, etc. Targets can also be referred to as anti-probes.

[0110] Wherein the probe binds to the target sequence, the binding can be "specific" or "selective". Generally, if the probe has one and only one binding partner (e.g., target), it has the property of "specificity". In fact, the vast majority of probes are "selective" rather than "specific" because most probes bind to many targets, especially at high concentrations. Therefore, these terms can be used interchangeably. The specificity and selectivity of binding can be determined using conventional methods. For example, where the target is a specific mRNA, the probe can be, for example, an oligonucleotide that specifically binds to the target under selected hybridization conditions without specifically binding to interfering RNA or DNA. Those skilled in the art can use methods recognized in the art to experimentally determine the characteristics of the oligonucleotide, which will hybridize best with the target and hybridize least with non-specific interfering DNA or RNA (e.g., see above). Typically, the length range of the oligonucleotide probe for distinguishing the target mRNA present in the background of a large excess of non-targeted RNA can be about 8 to about 50 nucleotides, preferably about 18, 20, 22 or 25 nucleotides. The oligonucleotide probe used in the biochemical assay without a large competitive target background can be shorter than 8 nucleotides. Using art-recognized programs (e.g., computer program BLAST), the sequences of the oligonucleotide probes can be selected so that they are unrelated to each other and different from potential interfering sequences in known genetic databases. Hybridization conditions that select to allow specific hybridization of the oligonucleotide probe to the RNA target can be routinely determined using art-recognized programs.

[0111] As used herein, the term "primer" refers to a short nucleic acid molecule, such as a DNA oligonucleotide comprising nine or more nucleotides, which in some instances is used to initiate the synthesis of a longer nucleic acid sequence. The length of a longer primer can be about 10, 12, 15, 20, 25, 30 or 50 nucleotides or longer. Primers can also be used for detection.

[0112] As used herein, "mechanically removing or ablating" certain samples means separating the sample from the substrate by mechanical means or vaporizing or decomposing the sample so that it is no longer present on the substrate.

[0113] Chemically "disintegrating" macromolecules herein refers to denaturing or disintegrating macromolecules, such as RNA, DNA and / or proteins, or chemically modifying them sufficiently so that they do not contaminate subsequent analysis of RNA, DNA and / or proteins in ROI tissue.

[0114] "Marks" made on slides by pathologists or laboratory users or other individuals are referred to herein as "manual marks". Such marks can be made by any available means, such as with a pen or etching device. In contrast, marks automatically made by the system herein may be referred to as "virtual marks" or "digital marks" to indicate that the marks are not made manually, but rather by using one or more algorithms.

[0115] The terms "digital," "digitized," "automated," and "automatic" and the like refer to operations performed by the systems herein, as opposed to operations performed manually by a user, such as controlled by an algorithm and / or through interaction between a user and a computer user interface.

[0116] As used herein, the term "surface" refers to any material that provides a site that allows interaction between analytes or target probes. Preferably, the surface is a solid support such as the surface of nitrocellulose, the wall of the hole of a reaction disk, a porous plate, a test tube, polystyrene beads, magnetic beads, membranes and microparticles (e.g., latex particles). The term contemplates suitable porous materials with sufficient porosity to allow detection reagents to approach and appropriate surface affinity to fix capture reagents (e.g., oligonucleotides). For example, the porous structure of nitrocellulose has excellent absorption and adsorption qualities for a variety of reagents (e.g., capture reagents). Nylon has similar properties and is also suitable. Microporous structures are useful, as are materials with gel structures in a hydrated state. Other examples of useful solid supports include natural polymeric carbohydrates and synthetically modified, cross-linked or substituted derivatives thereof, such as agar, agarose, cross-linked alginic acid, substituted and cross-linked guar gum, cellulose esters (especially cellulose esters with nitric acid and carboxylic acids), mixed cellulose esters, and cellulose ethers; nitrogen-containing natural polymers, such as proteins and derivatives, including cross-linked or modified gelatin; natural hydrocarbon polymers, such as latex and rubber; synthetic polymers that can be prepared with an appropriate porous structure, such as vinyl polymers, including polyethylene, polypropylene, polystyrene, polyvinyl chloride, polyvinyl acetate and partially hydrolyzed derivatives thereof, polyacrylamide , polymethacrylates, copolymers and terpolymers of the above condensation polymers (e.g. polyesters, polyamides) and other polymers (e.g. polyurethanes or polyepoxides); porous inorganic materials, such as sulfates or carbonates of alkaline earth metals and magnesium, including barium sulfate, calcium sulfate, calcium carbonate, silicates of alkali metals, alkaline earth metals, aluminum and magnesium; aluminum or silicon oxides or hydrates, such as clay, alumina, talc, kaolin, zeolites, silica gel or glass (these materials can be used as filters with the above polymer materials); and mixtures or copolymers of the above categories, such as graft copolymers obtained by initiating polymerization of synthetic polymers on existing natural polymers.

[0117] As used herein, the term "signature" refers to a collection of markers that indicate a target phenotype, for example, a cancer signature comprising ≥3 mutations, which indicates that the cell or tissue carrying the mutation is a tumor cell. In some embodiments, the feature includes the presence, absence, and / or abundance of a combination of markers such as tumor markers. By combining various probe sets, a reliable method for detecting a target phenotype can be designed. This feature test performed as a single assay can provide great benefits for evaluating and understanding the interactions between various markers.

[0118] The term "amplification" generally refers to the generation of multiple nucleic acid molecules from a target nucleic acid, wherein primers hybridize to specific sites on the target nucleic acid molecules to provide a starting site for extension by a polymerase. Amplification can be performed by any method generally known in the art, such as, but not limited to, standard PCR, long PCR, hot start PCR, qPCR, RT-PCR, and real-time PCR.

[0119] As used herein, the term "antibody" refers to an intact immunoglobulin, such as IgA, IgD, IgE, IgG or IgM, or an antibody fragment (particularly an antigen binding fragment), such as Fab, Fv or Fc, or a fusion antibody, a fusion antibody fragment or any other derivative of an antibody. The term "labeled antibody" refers to an antibody labeled with an enzyme, a fluorescent dye, a chemiluminescent substance, biotin, avidin or a radioactive isotope.

[0120] The term "epitope" refers to an antigenic region of a compound (e.g., a protein, carbohydrate, or lipid). The antigenic region is usually composed of 5 to 8 amino acids. The epitope is specifically recognized by the antigen binding site of the corresponding antibody.

[0121] As known to those skilled in the art, the term "fixed tissue or cells" is used herein and refers to biological tissues or cells that have been preserved from decay by chemical fixation methods. Such methods can prevent autolysis or corruption within such biological tissues or cells. Fixation can terminate biochemical reactions and increase the mechanical stability of the treated tissue.

[0122] The term "immunohistochemistry" or "IHC" refers to a technique for detecting the presence of an antigen in a histological sample using an antibody that is able to specifically bind to the antigen. Detection of the antibody-antigen complex is usually performed by a colorimetric reaction using an enzyme-labeled antibody or by a fluorescently labeled antibody.

[0123] As used herein, the term "macrodissection" refers to the process of scraping a target area from a tissue section mounted on a solid support such as a microscope slide by using a tool such as a cutting knife or a scraper. As used herein, the term "microdissection" refers to the process of cutting and separating one or more specific cells or target areas from a tissue sample. Microdissection can be performed, for example, using laser capture microdissection (LCM) by cutting the area of ​​interest with a laser.

[0124] As used herein, the term "membrane slide" refers to a solid support or microscope slide used for laser capture microdissection (LCM). For microdissection, a slide covered with a membrane or a frame slide consisting of a metal frame covered with various membranes can be used.

[0125] The term "polylysine" refers to a molecule comprising up to several hundred repeating units and is suitable for increasing the affinity between a sample (e.g., a tissue section) and the membrane side on which the sample is mounted. The polylysine according to the specification is poly-L-lysine. The poly-L-lysine according to the specification has a molecular weight of 70 to 300 kDa. Poly-L-lysine can be digested by proteases. Another polylysine according to the specification is, for example, poly-D-lysine. The poly-D-lysine according to the specification has a molecular weight of 70 to 300 kDa. Poly-D-lysine is resistant to protease digestion.

[0126] The term "qPCR" generally refers to a PCR technique known as real-time quantitative polymerase chain reaction, quantitative polymerase chain reaction, or kinetic polymerase chain reaction. This technique uses PCR to simultaneously amplify and quantify a target nucleic acid, where quantification is performed with the aid of an embedded fluorescent dye or sequence-specific probe containing a fluorescent reporter molecule that can only be detected after hybridization with the target nucleic acid.

[0127] As known to those skilled in the art, the term "RNA" is used herein and refers to mRNA precursors, mRNA precursor transcripts, mRNA, transcript processing intermediates, mature mRNA for translation, and transcripts from one or more genes, or nucleic acids derived therefrom. Transcript processing includes processes such as splicing, editing, modification, and degradation. The mRNA comprising a sample includes, for example, mRNA, mRNA transcripts of one or more genes, cDNA derived from mRNA using reverse transcription, RNA transcribed from amplified DNA, cRNA transcribed from cDNA, DNA amplified from a gene, and the like.

[0128] A "container" that may include a sample herein is broadly interpreted to mean a container of any type, shape, or size, including a surface, well, tube, or vial. A container need not be of any particular shape or size made of any particular material, but merely serves as a physical structure that enables analysis or manipulation of tissue located therein or thereon.

[0129] As used herein, a "stained" slide / substrate is a substrate that has been treated to help reveal the difference between the ROI sample and other samples so that a pathologist or other trained individual can mark the substrate to represent the outline of any ROI on the substrate. An "unstained" slide / substrate is a substrate that has not been treated but may or may not have been subjected to other types of treatments.

[0130] The term "separating" one or more ROIs from an X sample on a substrate includes methods in which the X sample is physically removed from the substrate, ablated (e.g., vaporized or burned or physically decomposed), or chemically treated so that it does not contaminate subsequent analysis of molecules in the ROI sample.

[0131] The term "substrate" herein refers to various slides, including but not limited to FFPE slides, tissue slides, standard slides, containers, stained slides, unstained slides, living tissues, etc.

[0132] The term "sample" as used herein refers to cell tissue, specimen, tissue sample, FFPE tissue, or other biological material fixed using standard molecular biology methods.

[0133] Herein, "computer processor" or "computing device" or "computer" is broadly interpreted as referring to any hardware and / or software combination that will perform its required functions. For example, the processor can be a programmable digital microprocessor, such as available in the form of an electronic controller, a mainframe, a server, or a personal computer (desktop or portable). In the case where the processor is programmable, appropriate programming can be communicated to the processor from a user interface incorporated in the computer body or located at a remote location, or can be pre-stored in a computer program product (such as a portable or fixed computer-readable storage medium, which can be based on magnetic, optical or solid-state devices). For example, a magnetic medium or an optical disk can carry programming and can be read by a suitable reader that communicates with each processor at its corresponding user interface. "User interface" herein is broadly interpreted as representing a physical structure that allows a user to program a computer and thereby control certain operations of the system through the computer. Examples include keyboards and monitors of mainframe or portable computers, other types of visual monitors and keyboard systems, such as tablet or smart phone devices or other remote devices. The user interface can be a physical part of the computer body, can be located elsewhere in the system, can be located at a remote location of the computer, and can communicate with the computer processor via a wired or wireless connection.

[0134] Method for digitally marking ROI on unstained substrate

[0135] Sample analysis and cutting typically involves a series of slides containing parallel sample sections. One or more slides in a set can be stained, for example, to reveal individual cell nuclei and / or to help distinguish different types of cells, such as cancer cells and non-cancerous cells in oncology applications. A pathologist can examine the substrate and mark the target region (ROI) on the slide with a pen or other suitable marking device. These ROIs or associated pen marks can then be positioned (rotated and aligned) with the substrate of adjacent slices of the sample and ultimately analyzed, such as extracting DNA for genome sequencing, extracting RNA for RNA expression analysis, or performing in situ analysis of cells, etc. In a manual sample cutting method, the pathologist's pen marks are manually transferred to the substrate, and then a razor blade is used to cut out the ROI from the surrounding sample on the slide.

[0136] Macrodissection techniques, which involve histological sectioning of not only the target region but also the surrounding tissue of the organ under investigation, have been increasingly used in many pathological studies, such as tumor typing, diagnosis of inflammatory diseases, and determination of degenerative diseases. In macrodissection, the target patient tissue ("S" region) is collected from a tissue sample (e.g., a slide), while unwanted regions ("X" regions) are excluded, so that only the "S" region is used as input material for downstream assays. In some cases, the complex "S" shape definition provided by the pathologist may result in difficult scraping operations for the histotechnician. Complex scraping techniques are often required, such as Figure 1 The present disclosure relates to systems and methods for processing biological samples so that analytes therein can be detected more accurately, preferably without interference from non-analytes, and thus, by improving signal quality and / or reducing noise, the systems and methods of the present invention greatly improve the results of biological assays, especially in the context of analyzing analytes in heterogeneous samples (e.g., FFPE).

[0137] By improving assay parameters, such as signal quality and reducing noise, the presently disclosed systems and methods also improve assay purposes, e.g., correlating the presence / absence or level or activity of a biomarker with a target characteristic, such as an ethnicity characteristic (for forensics) or a disease characteristic.

[0138] The systems and methods of the present disclosure are based in part on the use of a particle microinjector to target and remove specific regions of tissue in a biological sample (e.g., a histological section) and selectively collect tissue regions containing a signal ("S"). The method includes the use of particles and a controlled compressed gas or other gas flow to direct the particles to the ROI. The method is preferably a dry method, which reduces the chance of sample contamination and / or analyte dilution in the sample.

[0139] The currently disclosed method can be used in a variety of settings. For example, in a first implementation where macro-cutting is required, the "X" area in a substrate (e.g., a histological slide) can be selectively micro-blasted, leaving only the "S" area on the substrate unaffected. Afterwards, the "S" area can be removed from the substrate using a downstream processing method. In a second implementation where macro-cutting is also required, the "S" area in a substrate (e.g., a histological slide) can be selectively micro-blasted, leaving only the "X" area on the substrate unaffected. The tissue in the "S" area can be removed from the substrate, and then the particles can be collected in a container for processing using downstream processing. In another implementation where macro-cutting is not required, all areas of the substrate (the "X" and "S" areas) are micro-blasted, and the contents of these two areas are collected in a container for downstream processing.

[0140] Figure 2 An exemplary process for digitally processing unstained slide submission (USS) where quality and tissue thickness cannot be uniformly controlled is shown. The process may include two parallel processes with three paths that may result in parallel process merging.

[0141] According to one aspect, along the first parallel path, a USS can be mounted on a support. The mounted USS can be stored in a storage unit pending path inspection. A scanned image of the USS is created. The USS is retrieved from the storage unit. An automatic sample cutting procedure can then be performed.

[0142] According to one aspect, along a second parallel path, USS is stained to produce a reference slide. The reference slide is scanned to create an image. A digital markup is created that includes a region of interest (ROI) on the reference slide. It is then determined whether the markup can be uploaded to an image management system (IMS). If so, the second parallel path is merged with the first parallel path via path 1.

[0143] In Path 1, the presenting substrate successfully transfers its markers through current digital pathology solutions. USS can be processed earliest in current digital workflows through Path 1.

[0144] If not, an external marker transfer algorithm is applied. If the marker can be transferred, then Path 2 connects the second parallel path to the first parallel path. For example, in Path 2, presenting the substrate requires the development of a marker transfer algorithm external to the current digital pathology solution. This path assumes the need for an external marker transfer algorithm (e.g., a capability in the current digital pathology solution).

[0145] Finally, manual transfer of labels is possible via Path 3. In Path 3, for example, direct label transfer from the presenting substrate is not possible and a pathologist is required to manually perform digital labeling on each relevant substrate. This path is a fallback solution in the event that neither Path 1 nor Path 2 can proceed. The process can still at least be processed in an automated sample scraping system.

[0146] Figure 3 Methods of collecting the "S" area or removing the "X" area are shown. According to one aspect, the "X" area is removed and only the "S" area is left to allow a straight-through (collect all samples from the slide) method for sample collection. This is an effective method because there are usually only small "X" areas. For example, about 50% of the cases are straight-through (e.g., no "S" area). In most cases, the "X" area is much smaller than the "S" area (e.g., there is less "X" area to remove).

[0147] Other aspects include decomposing the nucleic acid in the "X" region (e.g., leaving only the "S" region and the inactive "X" region), thereby eliminating the need to remove the "X" region from the substrate. For example, the decomposed "X" region does not contain any quantifiable DNA / RNA remaining in the "X" region that further affects the analysis. Therefore, there will be no analyzable amount of DNA or RNA in the "X" region.

[0148] Other aspects include directly lysing samples on substrates as a straight-through method. This method can partially decompose the phospholipid bilayer of the cell and / or completely decompose the protein, thereby processing the sample into a liquid lysate suitable for downstream analytical processes. For example, the sample can be exposed to a lysis buffer or a buffer solution. Examples of lysates include: hypertonic, hypotonic, pH-adjusted solutions or solutions containing enzymes (e.g., proteases).

[0149] The described processes can be adapted to both automated and manual workflows. For example, they can be implemented in conjunction with current manual scraping techniques, since there are many more "S" regions than "X" regions, it is easier to remove or decompose the "X" regions than to collect only the "S" regions.

[0150] Sample cutting process

[0151] Figure 4 Various sample cutting methods are shown. According to aspects of the present disclosure, removal of the "X" region can leave only the "S" region on the slide for collection. For example, all unwanted sample on the substrate can be removed, leaving only the desired sample on the slide as a pass-through extraction. To remove the "X" region, mechanical and non-mechanical tools can be used to extract the unwanted sample from the substrate.

[0152] Mechanical (e.g., contact) tools may include the use of physical materials such as razor blades, machine milling (standard or custom milling tools), scrapers, punches, spoons, micro-bead blasting, and vacuum suction to remove unwanted sample directly from the substrate. Mechanical tools may also include particle blasting for removal of "X" areas.

[0153] Non-mechanical tools may include the use of indirect contact techniques, such as lasers and water jets, to remove unwanted samples directly from the substrate. Various laser systems include femtosecond laser systems, picosecond laser systems, nanosecond laser systems, microsecond laser systems, carbon dioxide laser systems, mode-locked laser systems, pulsed laser systems, Q-switched laser systems, Nd:YAG laser systems, continuous laser systems, dye laser systems, tunable laser systems, titanium sapphire laser systems, high-power diode laser systems, continuous wave lasers, or high-power fiber laser systems. For example, a femtosecond laser system may utilize an average power of 20 W, which is the size of a mid-tower personal computer (PC). Other examples of lasers include, but are not limited to, diode lasers, solid-state lasers, gas lasers, and dye lasers. It should be understood that the laser utilized may be configured to be lower in power to produce less footprint than conventional lasers.

[0154] Nucleic acids (e.g., DNA) may be degraded (e.g., denatured) in the "X" regions, leaving only the "S" regions on the slide and the inactive "X" regions for collection. For example, degrading nucleic acids in the "X" regions will render those regions inactive for subsequent processing (e.g., nucleic acid content is no longer at a level that affects gene expression). Thus, there is no need to physically remove / cut those regions, and a pass-through sample collection method can be used.

[0155] Non-mechanical methods such as laser, heat, RF, ultrasound, cryogenics, plasma, etc. can be used to decompose the unwanted "X" areas from the substrate (eg, a glass slide).

[0156] Chemical methods can include applying chemical agents (e.g., bleach, acids, bases, enzymes, etc.) to the "X" region to decompose nucleic acids (e.g., DNA / RNA). NaOH or salts can also be used to denature nucleic acids. Additional denaturants can include protein denaturants and nucleic acid denaturants. Exemplary concentrations of bleach can be at least 10% bleach. Combinations of chemical agents can be combined, including pH adjusted solutions containing endonucleases and / or proteases or 0.05%-10.0% (weight / volume) sodium hypochlorite.

[0157] According to various aspects, performing the lysis process directly on the substrate using a lysis buffer can bypass the sample cutting process. For example, the substrate with the sample can be directly immersed in a temperature-controlled container with a lysis buffer to separate the entire sample from the substrate. Subsequent protein kinase (ProK) protein digestion can be performed in the same container and in a separate process / container.

[0158] According to aspects of the present disclosure, sample cutting can include mechanical and non-mechanical methods to extract unwanted samples (e.g., unwanted "X" areas) from a substrate. As described above, mechanical (contact) methods can include using physical materials to cut unwanted samples directly from a substrate. The system can utilize a physical scraping tool (e.g., particle micro-sandblasting, sandblasting, razor blades, scrapers, punches, or spoons) in combination with a vacuum suction tube after the scraping action to simultaneously collect unwanted samples using a suction method.

[0159] The suction device is composed of low-cost disposable consumables (e.g., plastic tubes with filters) to collect all samples and will be replaced in each case to avoid cross contamination. Alternatively, the suction device can be cleaned between samples to eliminate cross contamination. The filter has the function of stopping the sample flow but allowing air to pass. In addition, the sample cannot completely block the filter to block air from passing.

[0160] The removed sample can then be collected in a waste bin, leaving only an "S" region on the substrate in a pass-through method. For example, pass-through collection can be achieved by: (a) direct single-pass razor blade pass-through, which can be easily automated; (b) utilizing a temperature-controlled ultrasonic water bath to separate the sample from the substrate into a container / tube; and / or (c) applying an electrostatic charge to the bottom of the collection container / tube after the sample has been separated from the substrate.

[0161] Non-mechanical methods may include the use of indirect techniques (e.g., such as laser and water jet ablation) to separate the unwanted sample from the substrate. For example, laser and water jet methods may include the use of high pressure water jets or laser ablation techniques to exert force on the target glass surface to separate the sample from the substrate. For the through method, this results in only an "S" region being left on the substrate.

[0162] Pass-through collection can be accomplished by: (a) direct single-pass razor blade pass-through, which can be easily automated; (b) separation of the sample from the substrate into a container / tube using a temperature-controlled ultrasonic water bath; and / or (c) application of an electrostatic charge to the bottom of the collection container / tube after the sample has been separated from the substrate.

[0163] In order to decompose the "X" region, the following methods can be used to extract the unwanted sample from the substrate. For example, non-mechanical methods can decompose the DNA in the unwanted "X" region from the substrate. According to one embodiment, the system will use non-contact ablation techniques, such as laser, heat, RF, ultrasound, cryogenics, or plasma, to decompose the unwanted sample from the substrate. For the straight-through method outlined above, this results in only the "S" region being left on the substrate.

[0164] Chemical methods can include applying chemicals to the "X" regions to break down the DNA. For example, the system can apply a chemical agent (such as bleach, acid, base, or enzyme, etc.) to the target "X" region on the glass surface to break down the sample from the substrate. For the straight-through method outlined above, this results in only the "S" region being left on the substrate.

[0165] It should be understood that the present disclosure is not limited to the field of pathology. It can also be used to pre-enrich or separate targets and non-targets. The determined technology can also be used alone or in combination with other integrated systems. It should also be understood that the method for direct flow can also include the use of pneumatics or a combination of the above methods.

[0166] Sample types include, but are not limited to, cell cultures, frozen sections, fresh samples, liquid biopsies, and cytology samples (ie, sputum, pleural fluid, etc.). Sample types may also include non-human targets.

[0167] The target sample is also not limited to a substrate. Any form of container can be used as an input to the system to allow the system to image the sample. Other examples include cover slips (i.e., to produce blood smears), bioreactors, cell culture dishes with imaging punches, sample collection paper, or liquid streams / droplets.

[0168] Aspects of the present disclosure provide advantages such as being clean, inexpensive, and compatible with high-throughput laboratories to manually extract target regions directly from substrates (e.g., using a pen), or providing easy-to-use digital annotation tools and object-based algorithms to match digital labels between substrates while maintaining the morphology of the sample, thereby eliminating the need to generate multiple sets of unstained substrates (USS) for manual microdissection. It also minimizes the risk of operator intervention required to complete the desired task.

[0169] In some embodiments, the cutting system can cut the substrate using a number of means such as lasers, water jets, and ultrasound to separate the ROI from the unwanted sample.

[0170] Some embodiments may include a low-cost mechanical system utilizing milling technology. A small desktop CNC milling machine uses a vertical end mill / scooper to collect predefined digital markers of ROI (sample to be saved) or RONI (sample to be discarded) samples. The desktop system allows the user to process a set of slides in a milling housing, and a vertical milling tool (e.g., a low-cost rotary scraping fixture with a suction feature) will be able to track and collect samples with predefined end-user digital annotations. In some embodiments, the preserved samples can be transferred to a tube for subsequent processing. In other embodiments, what remains on the substrate is an S sample, which can be easily and cleanly scraped with a razor blade.

[0171] For example, a large portion (e.g., more than 40%) of biopsy samples from certain cancers may be all S with no X, while many other samples have relatively small X regions. If a small end mill setup is used, the end mill may have an inner cavity for aspiration (i.e., the grinder may be a hypotube with or without a grinding tip to simplify machining of the sample). If the X region is small, the X region may be removed. If the S region is small, the S region may be machined and collected. For simplicity, it is also possible to uniformly remove all X regions or collect all S regions. In some embodiments, a water jet method may alternatively be used to remove the X sample on the slide, leaving the S sample.

[0172] In other embodiments, the X material is effectively ablated or destroyed on the slide, while the S material remains intact. This can be accomplished by various mechanical means, such as subjecting the X sample to various energy sources (e.g., particle microblasting (e.g., microbead blasting), laser, electrolysis, ultrasound, radiofrequency, or thermal energy sources) or by selective freeze drying of the X sample. In some embodiments, the means necessary to ablate the X sample include energy sources, such as laser, radiofrequency, current, sound, or thermal energy sources that are capable of lysing cells and / or decomposing biomacromolecules (e.g., nucleic acids and proteins). For example, some of these methods can effectively burn and vaporize the X sample. In some embodiments, the device can accurately direct the appropriate energy source to an area outside the pen or digital mark on the substrate, such as accurately focusing the laser or radiofrequency waves or ultrasound so that it affects only the X sample area. For example, in some embodiments, a pulsed laser, electrolysis, or ultrasound device can be used as a means to ablate and / or decompose nucleic acids in the X region of the slide, so that only the S region containing the ROI remains on the slide. For example, the system can be configured to direct the pulsed laser, electrolysis, or ultrasound device only to the X region once tracking of the S and X regions has been performed. For example, the slide can be scanned from only one end to the other with energy from a laser, electrolytic device, or ultrasound device, contacting only the X region of the slide, thereby ablating only the cells in the X region. As a result, the S region of the slide remains intact and is the only intact cellular material on the slide. In some embodiments, the energy can effectively vaporize the sample in the X region and can destroy target molecules, such as DNA and RNA, in those regions so that material from the X region does not contaminate the resulting isolated S region in subsequent analysis.

[0173] In other embodiments, the X region can be effectively removed by chemical treatment, including water cavitation methods (e.g., ultrasonic water cavitation methods), such as using one or more reagents that decompose molecules (e.g., DNA and / or RNA and / or protein) from the sample to be analyzed. Chemical means for selectively decomposing the X sample include, for example, adding bleach, strong acid, strong base, or enzymes (e.g., DNase, RNase, and protease) that target and decompose macromolecules. For example, based on the digital pen markings on the slide, the chemical treatment using RNase or protease can be directed only to the X region of the slide. RNase or protease can, for example, decompose RNA or protein into small fragments or monomers. As a result of the chemical treatment, for example, the X region of the slide will not contain a significant level of intact molecules for analysis, so, for example, the analysis of protein or RNA expression levels in the treated sample will only reflect the expression levels in the S portion of the sample, because only the protein and / or RNA from the S portion will remain intact.

[0174] Either of these two methods - ablating cells of the X portion to effectively remove the X tissue or chemically denaturing the X portion or the target molecule therein - can eliminate the need to collect the S tissue by cutting the S tissue from the slide containing the X and S tissues. Alternatively, in the "through" tissue collection method, the entire tissue on the slide can be collected or used for later analysis. For example, in the "through" collection, all tissues on the slide are removed, and there is no tissue cutting between the S and X regions. Therefore, the method herein is compatible with through tissue collection, and in the through tissue collection, when the tissue is collected for later analysis, it is not necessary to separate the S and X tissues on the slide. Alternatively, the tissue can be simply removed from the slide, for example, by scraping it off the slide and into a collection container by capillary action, suction, etc. with a rotating cutting tool similar to a milling process. In such an embodiment, even if the tissue collection is performed manually, the highly skilled razor blade technique currently used is not required, which can cause harm or lead to low accuracy. Alternatively, tissue collection can be performed automatically by an instrument. Such automation is more easily accomplished because the goal is to collect all tissue without separating region X from region S. For example, the tissue can be collected into a suitable container, such as a well, vial, or tube, for processing, such as cell lysis and extraction of target molecules (e.g., DNA, RNA, or protein). Means for mechanically collecting tissue from a processed slide into a container include, for example, sandblasting, scraping the tissue with a razor blade or similar blade, or suctioning the tissue with a spatula, spoon, punch, or vacuum, adding a solution or another substance to provide a competitive medium or surface compared to the slide surface, such as a charged surface or medium, etc.

[0175] In some embodiments, rather than collecting the tissue for analysis into a container such as a well, vial or tube, certain steps such as cell lysis may be performed directly on a slide, for example. Again, this may be possible when the X region is ablated or denatured to remove or inactivate any significant concentration of non-target molecules for later analysis. In some such embodiments, cell lysis may be performed directly on a tissue slide, for example using an appropriate kit, and optionally also extracting target molecules such as DNA, RNA or protein. In some embodiments, this process may be automatically controlled by an apparatus. For example, in some embodiments, a slide may be immersed in a lysis buffer such as contained in a well or tube or vial, so that cell lysis may be performed on a slide. In some embodiments, subsequent steps such as protease digestion or DNase digestion and / or RNA extraction may then be performed on the immersed slide.

[0176] Figure 5500 is a diagram of exemplary transillumination 506 and reflected illumination 504 of incident radiation 502 on mask 508. This is because radiation 502 is partially blocked by mask 508, and transillumination 506 can only reach certain areas of sample 512 (e.g., only a certain percentage of energy can be transmitted as a function of wavelength). Substrate 510 can be 1 mm thick, which provides some refraction to transillumination 506. For example, mask 508 can be on the top side of slide 510 so that radiation 502 is partially blocked by mask 508 and radiation can only reach "X" area. According to various aspects of the present disclosure, mask 508 can include optical, thermal, mechanical structures and / or chemical masks to block the relevant applied heat, laser, chemical agents (e.g., microbead blasting), etc. It should be understood that depending on the process used, the exposed portions can be removed or denatured.

[0177] Figure 6 An exemplary slide 600 is shown having marked unwanted "X" regions 602 and regions of interest (ROIs) 604 (eg, "S" regions) between the "X" regions 602. For example, the "X" regions 602 and the ROIs 604 may be determined by the process described above.

[0178] Figure 7 A process is shown for removing unwanted "X" regions 712 and 714 from a glass slide 720 by overlaying a mask 700 on the glass slide 720. For example, a masked glass slide 710 having depicted unwanted "X" regions 702, 704 and desired "S" regions 706 can be prepared. For example, the "X" regions 702, 704 and the "S" regions 706 can be determined by the above process. Overlaying the mask 700 on the glass slide 720 allows the unwanted "X" regions 712 and 714 to be removed from the glass slide 720 based on the depicted portions. The resulting glass slide 720 includes only the desired portion 716. It should be understood that, depending on the process employed, the "X" regions 712 and 714 can be removed or denatured.

[0179] Exemplary embodiments of automated tissue cutting (ATD) systems utilize a Fig.10 and Fig.11 Small milling system with one-off custom designed milling tool ( Fig.12 ), Fig.10 and Fig.11Depicts the distal cutting portion of a milling tool engaged with a tissue slide. At the front surface of the portion, there may be one or more cutting edges that will cut tissue from the slide during the rotation of the milling tool. The tool may have one or more lumens (i.e., channels) so that the excised tissue is pressed into the lumen by vacuum suction. The cutting portion may engage with the body of the tool. The function of the body is to hold the collected tissue, engage with the machine chuck, hold the filter element, and engage with the cutting portion. Compressed gas or other inert gases may be used to force the excised tissue into a collection tube. Alternative embodiments include stirring, transferring the rinse buffer to a collection tube by gravity, or placing the milling tool with the collected tissue directly into a test tube. In each case consisting of one or more tissue slides, a disposable milling tool may be used.

[0180] To reduce machining time, this custom milling cutter is designed to run at high speeds with direct drive or pneumatic operation. Rotational speeds can reach or exceed 100,000rpm. This allows for increased feed rates; therefore, the tool is designed to withstand the operating stresses and temperatures generated when running at high speeds.

[0181] The distal cutting region can have an inner cavity for collecting tissue ROI and have one or more cutting edges arranged radially on the front side. The material can be steel or any material that is harder than the tissue and facilitates low-cost high-volume processing. Engineering plastics may be a good choice because they can be injection molded economically. Since the ROI can be close to 750 microns or less for some tissue types, the diameter of the front side can be very small. A small inner cavity will seriously increase the pressure difference in the cutting section and restrict the airflow. It is preferred that the size of the inner cavity is rapidly reduced and immediately increased near the cutting interface.

[0182] The distal end of the body engages the cutting portion. The cutting portion can be threaded, insert molded, bonded, press-fitted, or any other suitable assembly technique. In one case, the interior space of the body is sufficient to accommodate tissue collected from all slides. The proximal end has features that engage with a standard machine chuck. The material can be steel or any material that maintains strength to withstand operating stresses and facilitates high-volume processing at low cost. Engineering plastics may be a good choice because they can be injection molded economically. The proximal end of the body also has internal features to engage with a filter. It also has suitable features to interface with a vacuum attachment, which can be an in-line or off-axis configuration driven by direct vacuum or by a Venturi device.

[0183] The filter element will block the passage of collected tissue, but allow air or inert gas to pass through. The filter opening size can be 50 microns or larger. The opening size and the overall size of the filter are designed so that the collected tissue will not completely block the filter at the end of the treatment. The filter needs to withstand the operating pressure and operating force. A pressure relief feature can also be integrated to prevent clogging. The filter can be bonded or mechanically attached to the body. Alternative clamping components can also be used.

[0184] The distal cutting portion of the milling tool is connected to the main body. The distal cutting portion may include one or more cutting edges engaged with a tissue slide. The distal cutting portion design directs the tissue being cut to the center of the cutting portion. One or more cutting edges at the distal position are radially oriented. Each cutting edge can be straight or curved. The contact angle of the blade with the ROI during operation makes it possible to generate a net vector force to push the tissue to the center of the treatment element, thereby allowing the collection of the ROI. For example, cutting edge #1 can be set to vertical, cutting edge #2 can form an acute angle, and cutting edge #3 can be set to an obtuse angle, each of these positions being set to drive the tissue being cut. A preferred embodiment is to set a distal cutting portion consisting of one or more blades being set at least like cutting edge #1, and a preferred direction is set to have an acute angle like cutting edge #2. The distal cutting portion can be designed to improve cutting ability, including the change of sawtooth edge and sawtooth position (including length, width and depth).

[0185] Fig.10 and Fig.11An exemplary sample milling device according to various embodiments is shown. As shown herein, the device 1000 includes a first component 1014 and a second component 1010. The first component 1014 has an opening on opposite ends, and the second component 1010 is fixed to one end of the first component 1014. The second component 1010 also includes a sample collection port 1018, which is fixed to the first component 1014 at a position facing away from the second component 1010, and the sample collection port 1018 has one or more sample scraping elements 1020 protruding along the periphery of the sample collection port 1018. The vacuum channel 1012 extends through the first component 1014 and the second component 1010 to connect the sample collection port 1018 with a vacuum connection port 1022 on the other end of the first component 1014. When the device 1000 is operated to collect a sample from a substrate, the device 1000 is rotated so that the sample scraping element 1020 mechanically removes a portion of the sample from the substrate surface, and at the same time, a vacuum is applied to the vacuum connection port 1022 of the first component 1014 using a vacuum pump, and the removed sample portion is collected through the sample collection port 1018 and the vacuum channel 1012. In various embodiments, the removed sample portion can be collected by the filter element 1016 connected to the vacuum connection port 1022. In various embodiments, the removed sample portion is collected in a container that is in fluid communication with the vacuum channel 1012.

[0186] In various embodiments, the first component 1014 and the second component 1010 are composed of different materials. In various embodiments, the first component 1014 and the second component 1010 are composed of the same material. Examples of materials that can be used include, but are not limited to: metal, polymer, fiberglass, etc.

[0187] In various embodiments, the first component 1014 and the second component 1010 are manufactured as a single integrated component, rather than two separate individual components.

[0188] In various embodiments, the sample scraping elements 1020 are evenly spaced along the perimeter of the sample collection port 1018. In various embodiments, the sample scraping elements 1104 are differently spaced along the perimeter of the sample collection port 1018.

[0189] Fig.12An exemplary sample collection system according to various embodiments is shown. As depicted herein, the system 1200 includes a sample milling device 1000 positioned above a substrate (i.e., a slide) 1202 holding a sample 1204. The sample milling device 1000 includes a first component 1014 and a second component 1010. When the sample milling device 1000 is operated, it is rotated and positioned (in the X, Y, and Z axes) so that the tip of the device 1000 contacts the sample 1204 to mechanically remove a portion of the sample 1204 from the surface of the substrate 1202 while collecting the removed portion of the sample by using a vacuum pump 1206 that applies a vacuum to an opening 1208 on one end of the milling device 1000.

[0190] In various embodiments, the sample milling device 100 can be moved in the X, Y, and Z directions so that it contacts only the desired portion of the sample 1204. In various embodiments, the slide 1202 holding the sample 1204 can be moved in the X, Y, and Z directions so that the sample milling device 1000.

[0191] In various embodiments, the first component 1014 and the second component 1010 are composed of different materials. In various embodiments, the first component 1014 and the second component 1010 are composed of the same material. Examples of materials that can be used include, but are not limited to: metal, polymer, fiberglass, etc.

[0192] Exemplary Subsequent Sample Analysis Procedure

[0193] The ROI tissue (or S tissue) can be collected so that it can be analyzed or manipulated in various ways. For example, in some embodiments, the collected ROI tissue is subjected to cell lysis as described above and then subjected to one or more other treatments. In some embodiments, DNA and / or RNA and / or proteins, cofactors, membrane lipids, etc. can be extracted from the ROI tissue directly or after cell lysis, or can be assessed in situ.

[0194] In some embodiments, the systems and methods herein relate to the analysis of DNA in ROI tissues. For example, the systems and methods herein can be used in conjunction with the following modes of ROI tissue analysis: copy number variation (CNV), single nucleotide polymorphism (SNP), point mutations in specific genes, detection of deletion or insertion mutations in genes, detection of transposition, translocation, the presence of foreign DNA (e.g., viral or bacterial DNA), methylation of DNA, etc. In some embodiments, the systems and methods herein can be used in conjunction with the analysis of RNA species in ROI tissues, such as determining the level of a specific RNA transcript of a gene or detecting a specific alternative splicing RNA transcript and its relative level, or the presence of interfering RNA. RNA analysis can be performed, for example, by methods including reverse transcription polymerase chain reaction (RT-PCR) (e.g., quantitative RT-PCR), or by whole transcriptome sequencing methods. In some embodiments, the presence or level of a specific protein in ROI tissues can be assessed, such as in situ or after a cell lysis procedure, by methods such as immunoprecipitation, ELISA, Western blotting, nucleic acids, etc. In some embodiments, the ROI tissue can be evaluated to detect the presence or levels of other molecules (eg, biological co-factors, cell membrane lipids or other components, etc.).

[0195] exist Fig. 8A and Figure 8B Exemplary systems and methods for analyzing biological samples such as FFPE tissues mounted on slides are shown in FIG.

[0196] Fig. 8AAn exemplary method for processing and / or analyzing a patient sample (e.g., a tumor biopsy sample) according to the above method is shown. A biological sample is micro-sandblasted with a contact medium comprising particles and air. In some embodiments, the contact medium may comprise particles that are very suitable for separating specific target analytes present in a patient sample. For example, silica particles or silica-coated particles, such as silica-coated ferromagnetic beads, are very suitable for separating nucleic acid markers, such as mRNA with missense mutations or loss-of-function mutations. The particle micro-injector is loaded with such particles, and the nozzle of the micro-injector is directed to a desired tissue region. For example, in the case of a pathological sample, the desired region may be a region containing nucleic acids, such as a cell nucleus stained with an appropriate stain. Alternatively, in the case of a patient sample containing different tissue types, the desired region may be a tissue type or tissue layer containing target cells, such as, in the case of pancreatic cancer, epithelial cells lining the pancreatic duct. The target region is micro-sandblasted with a contact medium (containing compressed gas and particles) to remove the target cells, which are then collected and merged with a tissue lysis buffer to destroy the cells. This produces a cell lysate solution in which the target analyte (e.g., in the case of pancreatic cancer, mutant nucleic acids encoding KRAS, TP53, CDKN2A, SMAD4, BRCA1 and / or BRCA2; see Cicenas et al., Cancers (Basel), 28, 9(5), 2017) is adsorbed onto the particles. To increase the rate of adsorption, the pH of the buffer solution can be adjusted to a pKa or below the pKa of the surface silanol groups in the silica particles, and the salt content of the buffer can be increased. The particles are then washed with a solution to adsorb the analyte on their surface while washing away other components of the lysate solution, such as non-analytes, such as proteins and lipids. The analyte can be directly analyzed using downstream analytical techniques such as PCR. Alternatively, the nucleic acids adsorbed on the surface of the silica particles are eluted with a suitable eluent prior to downstream analysis using a nucleic acid detection technique such as PCR. Elution can be facilitated using a buffer with low ionic strength and low pH. If desired, prior to microblasting, the sample can be pre-processed using a microdissection technique (e.g., laser microdissection) to further refine and / or target a region of interest (ROI).

[0197] Figure 8BAn exemplary method for processing and / or analyzing biological samples (e.g., fixed entomological samples for museum archives or contact slides containing soil microorganisms) according to the above method is shown. The biological sample is microblasted with a contact medium containing particles and gas. In some embodiments, the contact medium can contain particles that are very suitable for separating specific target analytes present in patient samples. For example, aluminum particles functionalized with amino, carboxyl, sulfonate and phosphate groups can be used to separate specific polypeptide markers. A particle microinjector is loaded with such particles, and the nozzle of the microinjector is directed to a desired tissue area. For example, in the case of a fixed insect sample, the desired area can be an area containing a target marker, such as an abdomen. The target area is microblasted with a contact medium (containing compressed gas and particles) to remove target cells, which are then collected and merged with a tissue lysis buffer to destroy the cells. This produces a cell lysate solution in which the target analyte is adsorbed onto the particles (e.g., peptides are adsorbed onto functionalized aluminum particles). To increase the rate of adsorption, the particles can be derivatized, depending on the physicochemical properties of the target (e.g., hydrophilicity in the case of soluble proteins; hydrophobicity in the case of membrane proteins). The particles are then washed with a solution so that the analyte is adsorbed on its surface while washing away other components of the lysate solution, such as non-analytes, such as lipids. The analyte can be directly analyzed using downstream analytical techniques such as mass spectrometry. Alternatively, the polypeptide adsorbed on the surface of the aluminum particles is eluted with a suitable eluent before downstream analysis using peptide detection techniques such as immunoblotting or mass spectrometry. If necessary, the sample can be pre-treated using a microdissection technique (e.g., laser microdissection) before microblasting to further improve and / or target the ROI.

[0198] Computer-implemented systems

[0199] Fig. 94 is a block diagram illustrating a computer system 400 on which embodiments of the present teachings may be implemented. In various embodiments of the present teachings, the computer system 400 may include a bus 402 or other communication mechanism for communicating information, and a processor 404 coupled to the bus 402 for processing information. In various embodiments, the computer system 400 may also include a memory, which may be a random access memory (RAM) 406 or other dynamic storage device, coupled to the bus 402 to determine instructions for execution by the processor 404. The memory may also be used to store temporary variables or other intermediate information during the execution of instructions executed by the processor 404. In various embodiments, the computer system 400 may also include a read-only memory (ROM) 408 or other static storage device coupled to the bus 402 for storing static information and instructions for the processor 404. A storage device 410, such as a magnetic disk or optical disk, may be provided and coupled to the bus 402 to store information and instructions.

[0200] In various embodiments, the computer system 400 can be coupled to a display 412, such as a cathode ray tube (CRT) or a liquid crystal display (LCD), via the bus 402 to display information to a computer user. An input device 414 including alphanumeric keys and other keys can be coupled to the bus 402 to communicate information and command selections to the processor 404. Another type of user input device is a cursor control 416, such as a mouse, trackball, or cursor direction keys, for communicating direction information and command selections to the processor 404 and controlling cursor movement on the display 412. This input device 414 typically has two degrees of freedom in two axes, a first axis (i.e., x) and a second axis (i.e., y), allowing the device to specify a position in a plane. However, it should be understood that input devices 414 that allow 3-dimensional (x, y, and z) cursor movement are also contemplated herein.

[0201] Consistent with certain embodiments of the present teachings, in response to processor 404 executing one or more sequences of one or more instructions contained in memory 406, computer system 400 can provide results. Such instructions can be read into memory 406 from another computer-readable medium or computer-readable storage medium (e.g., storage device 410). Execution of the sequences of instructions contained in memory 406 can cause processor 404 to perform the processes described herein. Alternatively, hard-wired circuits can be used in place of or in combination with software instructions to implement the present teachings. Therefore, implementation of the present teachings is not limited to any specific combination of hardware circuitry and software.

[0202] As used herein, the term "computer-readable medium" (e.g., data storage, data storage, etc.) or "computer-readable storage medium" refers to any medium that participates in providing instructions to processor 404 for execution. Such media can take many forms, including but not limited to non-volatile media, volatile media, and transmission media. Examples of non-volatile media can include but are not limited to optical, solid-state, magnetic disks, such as storage device 410. Examples of volatile media can include but are not limited to dynamic memory, such as memory 406. Examples of transmission media can include but are not limited to coaxial cables, copper wire, and optical fiber, including the wires that comprise bus 402.

[0203] Common forms of computer readable media include, for example, floppy disks, diskettes, hard disks, magnetic tape or any other magnetic medium, CD-ROMs, any other optical medium, punch cards, paper tape, any other physical medium with a pattern of holes, RAM, PROM and EPROM, FLASH-EPROM, any other memory chip or tape, or any other tangible medium from which a computer can read.

[0204] In addition to computer-readable media, instructions or data can also be provided as signals on a transmission medium included in a communication device or system to provide a sequence of one or more instructions to the processor 404 of the computer system 400 for execution. For example, a communication device may include a transceiver with a signal indicating instructions and data. The instructions and data are configured to enable one or more processors to implement the functions outlined in this disclosure. Representative examples of data communication transmission connections may include, but are not limited to, telephone modem connections, wide area networks (WANs), local area networks (LANs), infrared data connections, NFC connections, etc.

[0205] It should be understood that the flowcharts, diagrams, and accompanying disclosed methods described herein may be implemented using computer system 400 as a standalone device or on a distributed network of shared computer processing resources, such as a cloud computing network.

[0206] Depending on the application, the methods described herein can be implemented by various means. For example, the methods can be implemented in hardware, firmware, software, or any combination thereof. For hardware implementation, the processing unit can be implemented in one or more application specific integrated circuits (ASICs), digital signal processors (DSPs), digital signal processing devices (DSPDs), programmable logic devices (PLDs), field programmable gate arrays (FPGAs), processors, controllers, microcontrollers, microprocessors, electronic devices, other electronic units designed to perform the functions described herein, or a combination thereof.

[0207] In various embodiments, the methods of the present teachings may be implemented as firmware and / or software programs and applications written in conventional programming languages ​​such as C, C++, Python, etc. If implemented as firmware and / or software, the described embodiments may be implemented on a non-transitory computer-readable medium having stored thereon a program for causing a computer to perform the above methods. It should be understood that the methods may be implemented in a computer such as Fig. 9 The various engines described herein are provided on a computer system such as computer system 400, whereby processor 404 will perform the analyses and determinations provided by these engines based on instructions provided by any one or a combination of storage components 406 / 408 / 410 and user input provided through input device 414.

[0208] Although the present teaching is described in conjunction with various embodiments, it is not intended that the present teaching be limited to such embodiments. On the contrary, as will be appreciated by those skilled in the art, the present teaching encompasses various alternatives, modifications and equivalents.

[0209] In addition, when describing various embodiments, the specification may have proposed a method and / or process as a specific order of steps. However, to the extent that the method or process does not rely on the specific order of steps set forth herein, the method or process should not be limited to the specific order of steps described. As will be appreciated by those of ordinary skill in the art, other orders of steps are possible. Therefore, the specific order of steps set forth in the specification should not be interpreted as a limitation on the claims. In addition, the claims for the method and / or process should not be limited to performing its steps in the order written, and those skilled in the art can easily understand that the order can be changed and still be within the spirit and scope of the various embodiments.

[0210] The embodiments described herein may be implemented with other computer system configurations including handheld devices, microprocessor systems, microprocessor-based or programmable consumer electronics, minicomputers, mainframe computers, etc. The embodiments may also be practiced in distributed computing environments where tasks are performed by remote processing devices that are linked through a network.

[0211] It should also be understood that the embodiments described herein may employ various computer-implemented operations involving data stored in a computer system. These operations are operations that require physical manipulation of physical quantities. Typically, although not necessarily, these quantities take the form of electrical or magnetic signals that can be stored, transmitted, combined, compared, and otherwise manipulated. In addition, the operations performed are typically referred to in the following terms, such as generating, identifying, determining, or comparing.

[0212] Any operation that forms part of the embodiments described herein is a useful machine operation. The embodiments described herein also relate to equipment or devices for performing these operations. The systems and methods described herein may be specifically constructed for the desired purpose, or it may be a general-purpose computer selectively activated or configured by a computer program stored in the computer. In particular, various general-purpose machines may be used together with computer programs written according to the teachings of this article, or it may be more convenient to construct more specialized devices to perform the desired operations.

[0213] Certain embodiments may also be embodied as computer readable code on a computer readable medium. The computer readable medium is any data storage device that can store data that can then be read by a computer system. Examples of computer readable media include hard drives, network attached storage (NAS), read-only memories, random access memories, CD-ROMs, CD-Rs, CD-RWs, magnetic tapes, and other optical, flash memory, and non-optical data storage devices. Computer readable media may also be distributed on network coupled computer systems to store and execute computer readable code in a distributed manner.

[0214] Example

[0215] The structures, materials, compositions and methods described herein are intended to be representative examples of the present disclosure, and it will be understood that the scope of the present disclosure is not limited to the scope of the examples. Those skilled in the art will recognize that the present disclosure can be practiced by modifying the disclosed structures, materials, compositions and methods, and such modifications are considered to be within the scope of the present disclosure.

[0216] Example 1: Processing of biological samples to obtain nucleic acid analytes

[0217] The histological sample containing the target nucleic acid is processed according to the above method. Here, the contact medium comprises particles that are very suitable for nucleic acid separation. These particles include silica particles or silica-coated particles, such as silica-coated ferromagnetic beads. The particle microinjector is equipped with silica particles to provide an effective method for separating nucleic acids from tissue samples. First, the required tissue area is removed from the slide by micro-sandblasting using silica particles, which is then collected and merged with tissue lysis buffer to destroy cells. This produces a cell lysate solution, in which nucleic acids are adsorbed onto the silica particles. The silica particles are then washed with a solution that adsorbs nucleic acids on their surface while washing away other components of the lysate solution, such as proteins and lipids. Downstream analysis techniques such as PCR can be used to directly analyze nucleic acids. Alternatively, before downstream analysis is performed using nucleic acid detection techniques such as PCR, the nucleic acids adsorbed on the surface of the silica particles are eluted with a suitable eluent. If necessary, microdissection techniques (such as laser microdissection) can be used to pre-treat the sample.

[0218] Example 2: Processing of biological samples to obtain nucleic acid and peptide analytes

[0219] Alternatively, the particles used for micro-sandblasting in the above-mentioned embodiment 1 are collected in a container and merged with other particles that are most suitable for selectively binding various potential analytes (such as nucleic acids or proteins), and the mixture of the obtained particles is used to separate the target analyte. As in Example 1, in this alternative setting, the micro-sandblasting particles that are combined with the target analyte (for example, based on charge or hydrophobicity or affinity) are first selected. The mixture of tissue and particles produced after micro-sandblasting is merged with lysis buffer to destroy cells, and then the resulting lysate solution is merged with the particles with oligonucleotides or antibodies on the surface, wherein the oligonucleotides or antibodies specifically bind the target analyte. Then the particles are directly analyzed (such as chromatography or spectroscopy). Alternatively, these particles are subjected to a series of combinations, washing and elution steps to elute the target analyte, and then analyzed using conventional techniques. Finally, when the target analyte is a protein, the lysate solution can also be subjected to other analytical steps, such as enzyme assays, binding assays, functional assays, etc.

[0220] The above method can be combined with any separation and / or purification steps, which can be performed at any stage of the workflow, preferably before the final analysis step such as PCR (in the case of nucleic acid analytes) or ELISA (in the case of protein analytes). For example, the target nucleic acid is usually separated from the tissue by a series of steps, the steps including: (a) tissue lysis, in which the cells of the tissue are destroyed by various methods to break the cells open and release their contents into the solution; (b) the nucleic acid is adsorbed onto the surface of a solid phase; (c) the solid phase is washed with a solution to adsorb the nucleic acid on the solid phase, but other biomolecules are removed; and (d) the solid phase is washed with a solution to elute the nucleic acid from the solid phase so that the collected eluate contains the separated nucleic acid. In this method, silica membranes and silica particles are usually used as solid phases. Other types of particles are also commonly used, including silica-coated or polymer-coated ferromagnetic beads.

[0221] From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of the method, and without departing from the spirit and scope thereof, can make various changes and modifications to adapt it to various usages and conditions.

[0222] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those of ordinary skill in the art to which the present disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described in the preceding paragraphs. In addition, materials, methods and embodiments are illustrative only and are not intended to be restrictive. In the event of a conflict, the present specification including definitions shall prevail.

[0223] All U.S. patents and published or unpublished U.S. patent applications cited herein are incorporated herein by reference. All disclosed foreign patents and patent applications cited herein are incorporated herein by reference. All disclosed references, documents, manuscripts, scientific literature cited herein are incorporated herein by reference. All identifiers and accession numbers associated with scientific databases (e.g., PUBMED, NCBI) cited herein are incorporated herein by reference.

[0224] Description of selected implementation plans

[0225] Embodiment 1. A system for processing a sample fixed on a substrate, comprising: a support unit for fixing the substrate; a camera positioned near the support unit; a processing element configured to remove a portion of the sample fixed on the substrate; and a computing device communicatively connected to the support unit, the camera and the processing element, comprising an image capture engine configured to use a camera to obtain a first image of a first substrate having a first fixed sample and a second image of a second substrate having a second fixed sample; a digital marking engine configured to allow a user to generate a marked image containing a digital outline of the first image and a portion of the first fixed sample; an image overlay engine configured to overlay the marked image onto the second image to align the image outlines of the first fixed sample and the second fixed sample; and a sample removal engine configured to control the position of the support unit and the processing element to remove only a portion of the second fixed sample located in the digital outline of the first fixed sample.

[0226] Embodiment 2. A system according to embodiment 1, wherein the processing element is configured to remove a portion of the sample using a mechanical tool.

[0227] Embodiment 3. A system according to embodiment 2, wherein the mechanical tool includes at least one of a sandblast, a razor blade, a milling tool, a scraper, a punch, a spoon, and a vacuum.

[0228] Embodiment 4. A system according to any one of embodiments 1 to 3, wherein the processing element is configured to remove the portion of the sample using a non-mechanical tool.

[0229] Embodiment 5. A system according to embodiment 4, wherein the non-mechanical tool includes at least one of a laser or a water jet.

[0230] Embodiment 6. A method for processing a sample fixed on a substrate, comprising: obtaining a first image of a first substrate having a first fixed sample; obtaining a second image of a second substrate having a second fixed sample; generating a marked image including a digital outline of the first image and a portion of the first fixed sample; superimposing the marked image on the second image to align the image outlines of the first fixed sample and the second fixed sample; and using a processing element to remove only a portion of the second fixed sample that is located in the digital outline of the first fixed sample.

[0231] Embodiment 7. A method according to embodiment 6, wherein the removing includes removing the portion of the second fixed sample using a mechanical tool.

[0232] Embodiment 8. The method according to embodiment 6 or embodiment 7, further comprising: removing the portion of the second fixed sample using at least one of sandblasting, a razor blade, a scraper, a punch, a spoon, a vacuum, or a combination thereof.

[0233] Embodiment 9. A method according to embodiment 6, wherein the removing includes removing the portion of the second fixed sample using a non-mechanical tool.

[0234] Embodiment 10. The method according to embodiment 6 or embodiment 9, further comprising removing the portion of the second fixed sample using at least one of a laser or a water jet.

[0235] Embodiment 11. A system for processing a sample fixed on a substrate, comprising: a support unit for fixing a substrate; a camera positioned near the support unit; a processing element configured to provide a nucleic acid denaturing agent to denature nucleic acids on a portion of the sample fixed on the substrate; and a computing device communicatively connected to the support unit, the camera and the processing element, including an image capture engine configured to use a camera to obtain a first image of a first substrate having a first fixed sample and a second image of a second substrate having a second fixed sample; a digital marking engine configured to allow a user to generate a marked image containing the first image and a digital outline of a portion of the first fixed sample; an image overlay engine configured to overlay the marked image onto the second image to align the image outlines of the first fixed sample and the second fixed sample; and a nucleic acid denaturation engine configured to control the position of the support unit and the processing element so as to denature only the nucleic acids in a portion of the second fixed sample located in the digital outline of the first fixed sample, the nucleic acid denaturation engine comprising a chemical analyzer for performing chemical analysis, a mass spectrometer and / or a cell analyzer for performing cell analysis.

[0236] Embodiment 12. A system as described in Embodiment 11, wherein the nucleic acid denaturing agent comprises a chemical reagent.

[0237] Embodiment 13. A system according to embodiment 12, wherein the chemical agent includes at least one of bleach, acid, base or enzyme.

[0238] Embodiment 14. A system according to any one of embodiments 11 to 13, wherein the processing element is configured to remove the portion of the sample using a non-chemical tool.

[0239] Embodiment 15. A system according to embodiment 14, wherein the non-chemical tool includes at least one of a laser, a thermal heater, a radio frequency (RF) wave, ultrasound, low temperature, or plasma.

[0240] Embodiment 16: A method for processing a sample fixed on a substrate, comprising: obtaining a first image of a first substrate having a first fixed sample; obtaining a second image of a second substrate having a second fixed sample; generating a labeled image comprising the first image and a digital outline of a portion of the first fixed sample; superimposing the labeled image on the second image to align the image outlines of the first fixed sample and the second fixed sample; and using a processing element to denature nucleic acids in a portion of the second fixed sample that is only located in the digital outline of the first fixed sample.

[0241] Embodiment 17. A method according to embodiment 16, wherein the denaturation includes exposing the portion of the second fixed sample to a chemical reagent.

[0242] Embodiment 18: The method of Embodiment 16 or Embodiment 17, further comprising exposing the portion of the second fixed sample to at least one of a bleach, an acid, a base, or an enzyme.

[0243] Embodiment 19. The method of embodiment 16, wherein the denaturing comprises exposing the portion of the second fixed sample to a non-chemical means.

[0244] Embodiment 20. The method of embodiment 16 or embodiment 19, further comprising exposing the portion of the second fixed sample to at least one of a laser, a thermal heater, radio frequency (RF) waves, ultrasound, cryogenics, or plasma.

[0245] Embodiment 21: A sample processing device comprises: a first component having openings on opposite ends; a second component fixed to one end of the first component, wherein the second component also includes a sample collection port, the sample collection port being fixed to the first component at a position facing away from the second component, the sample collection port having one or more sample scraping elements protruding along the periphery of the sample collection port; a vacuum channel extending through the first and second components to connect the sample collection port with a vacuum connection port on the other end of the first component.

[0246] Embodiment 22. A sample processing device according to embodiment 21, wherein the first component and the second component are composed of different materials.

[0247] Embodiment 23. A sample processing device according to embodiment 22, wherein the first component and the second component are composed of the same material.

[0248] Embodiment 24: The sample processing device of any one of Embodiments 21 to 23, wherein the sample scraping elements are evenly spaced along the perimeter of the sample collection port.

[0249] Embodiment 25: The sample processing device of any one of Embodiments 21 to 23, wherein the sample scraping elements are differently spaced along the perimeter of the sample collection port.

[0250] Embodiment 26. The sample processing device of any one of Embodiments 21 to 25, further comprising a filter attached to the opening on one end of the first component.

[0251] Embodiment 27. A sample processing device according to embodiment 21, wherein the first component and the second component are manufactured as a single integrated device.

Claims

1. A method for processing a biological sample for use in a biological assay, comprising (a) contacting the biological sample with a contact medium comprising particulate matter blasted with compressed gas under conditions sufficient to achieve blasting of a non-target region (RONI) in the biological sample with the blasted particulate matter such that the RONI is at least partially transferred to the contact medium, wherein: The contacting includes selectively contacting the RONI in the biological sample with a contact medium while leaving a region of interest (ROI) in the biological sample uncontacted; (b) removing the contact medium from the biological sample; and (c) Cutting ROI or collecting ROI.

2. The method according to claim 1, wherein The biological sample is processed for analysis of one or more analytes of diagnostic interest.

3. The method according to claim 1, wherein The biological sample includes punch biopsy specimens, needle biopsy specimens, fresh tissue, tissue culture, frozen tissue specimens, neutral formalin treated tissue, organs, organelles, formalin fixed paraffin embedded (FFPE) tissue, ethanol fixed paraffin embedded (EFPE) tissue, hematoxylin and eosin (H&E) stained tissue or glutaraldehyde fixed tissue.

4. The method of claim 1 , wherein the biological sample comprises an analyte of diagnostic interest, the analyte being selected from genomic DNA (gDNA), methylated DNA, specific methylated DNA, messenger RNA (mRNA), fragmented DNA, fragmented RNA, fragmented mRNA, mitochondrial DNA (mtDNA), chloroplast DNA (ctDNA), viral RNA or viral DNA, microRNA, ribosomal RNA, in situ PCR products, polyA mRNA, RNA / DNA hybrids, lipids, carbohydrates, proteins, glycoproteins, lipoproteins, phosphoproteins, specific phosphorylated or acetylated variants of proteins, viral coat proteins, or nucleic acids.

5. The method according to claim 1, wherein The particulate matter in the blasting media includes aluminum oxide; silicon dioxide; metal-based particles; magnetic or ferromagnetic particles or combinations thereof.

6. The method according to claim 1, wherein The particulate matter is capable of binding to an analyte or non-analyte in a biological sample through an interaction selected from ionic interactions, polar-nonpolar interactions, hydrophobic interactions, van der Waals interactions, chemical coupling, dielectric or zwitterionic interactions, or combinations thereof.

7. The method according to claim 1, wherein The compressed gas is selected from compressed helium, argon, xenon, nitrogen, carbon dioxide or a combination thereof.

8. The method according to claim 1, wherein The biological sample is mounted on a substrate.

9. The method according to claim 1, wherein: The biological sample includes a nucleic acid analyte, and the particulate matter comprises silicon dioxide.

10. The method of claim 1, wherein the cutting is microdissection.

11. The method according to claim 1, wherein The contact medium is removed from the biological sample by vacuum treatment, pressure differential or gradient, gravity, a transport medium comprising a liquid, aerosol or gas, or a transfer medium selected from a magnetic field or an electric field.

12. The method according to claim 1, comprising: collecting particulate matter from the contact medium; preparing particulate matter for analysis; Analyze particulate matter.

13. The method according to claim 12, wherein: Preparing the particulate matter for analysis includes treating the particulate matter with a buffer and washing the particulate matter to remove non-analytes.

14. The method of claim 12, wherein the analysis of the particulate matter comprises polymerase chain reaction (PCR), quantitative PCR (qPCR), reverse transcriptase PCR (RT-PCR), nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP), rolling circle amplification (RCA), immunoassay, immunoPCR (iPCR), enzyme activity assay, staining, imaging, whole genome amplification (WGA), in situ PCR, in situ WGA, colony formation, sequencing, single molecule sequencing, nanopore analysis, nanopore sequencing, single molecule imaging, DNA sphere formation, electrophoresis, microelectromechanical system (MEMS) electrophoresis, mass spectrometry, chromatography, proximity ligation assay, electrochemical detection, plasmon resonance (SPR), hybridization assay, FRET, cell sorting, electrochemiluminescent ELISA, or chemiluminescent ELISA.

15. The method according to claim 1, wherein The biological sample comprises a two-dimensional tissue or a three-dimensional tissue comprising a well-defined spatial location of a region of interest (ROI), a region of non-target (RONI), or both a ROI and a RONI.

16. A method for determining an analyte in a biological sample, the method comprising treating the biological sample by: (a) contacting the biological sample with a contact medium comprising particulate matter blasted with compressed gas under conditions sufficient to achieve blasting of a non-target region (RONI) in the biological sample with the blasted particulate matter such that the RONI is at least partially transferred to the contact medium, wherein: The contacting includes selectively contacting the RONI in the biological sample with a contact medium while leaving a region of interest (ROI) in the biological sample uncontacted; (b) removing the contact medium from the biological sample; (c) cutting the ROI or collecting the ROI; and (d) Analysis of analytes in the ROI.

17. The method of claim 16, wherein the particulate matter comprises alumina; silica; metal-based particles; magnetic or ferromagnetic particles, or a combination thereof.

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