阅读说明：本技术 分析前基底处理的系统和方法 (System and method for analyzing pre-substrate processing ) 是由 杨金龙 袁谢安玫 李振威 加百列·耶稣·塞缪尔·佩拉斯·莫拉莱达 乔纳森·M·卡塞尔 于 2019-06-21 设计创作，主要内容包括：本公开中提出的一些实施方案涉及自动组织切割(ATD)系统。ATD系统是一站式且可能是低成本的系统,其可使用非接触和/或机械方法从基底上的病理学家数字标记或笔标记对基底进行切割,以提取福尔马林固定的石蜡包埋的(FFPE)组织样品：(a)仅将一个或多个ROI作为要保存的区域；(b)去除或分解非目标区域(RONI)中的核酸内容物,并将所有组织样品从标准显微镜基底收集到特定容器中。(Some embodiments presented in the present disclosure relate to an automated tissue cutting (ATD) system. The ATD system is a one-station and potentially low-cost system that can use non-contact and/or mechanical methods to dissect the fundus from the pathologist's digital or pen marks on the fundus to extract formalin-fixed paraffin-embedded (FFPE) tissue samples: (a) only one or more ROIs are taken as regions to be saved; (b) the nucleic acid content in the non-target Region (RONI) is removed or lysed and all tissue samples are collected from a standard microscope substrate into a specific container.)

1. A method of processing a biological sample for biological assays comprising (a) contacting the biological sample with a contact medium comprising particulate matter and a compressed gas under conditions sufficient to effect at least partial transfer of components in the biological sample to the contact medium; and (b) removing the contact medium from the biological sample.

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

3. The method of claim 1, wherein the biological sample comprises a punch biopsy sample, a needle biopsy sample, fresh tissue, a tissue culture, a frozen tissue sample, neutral formalin treated tissue, an organ, an organelle, 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, comprising contacting a region of interest (ROI), a non-region of interest (RONI), or all regions in the biological sample with the contact medium; preferably, a target Region (ROI) is contacted with the contact medium.

5. The method of claim 1, wherein the biological sample comprises at least one analyte of diagnostic interest 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, polyAmRNA, RNA/DNA hybrids, lipids, carbohydrates, proteins, glycoproteins, lipoproteins, phosphoproteins, specific phosphorylated or acetylated variants of proteins, or viral coat proteins; preferably a nucleic acid selected from mRNA, gDNA, viral DNA or viral RNA.

6. The method of claim 1, wherein the particulate matter in the blasting media comprises alumina; silicon dioxide; a metal-based particle; magnetic or ferromagnetic particles or combinations thereof.

7. The method of any one of claims 6, wherein the particulate matter is capable of binding an analyte or a non-analyte in a biological sample; preferably, the particulate matter is capable of binding the analyte via an interaction selected from ionic interactions, polar-non-polar interactions, hydrophobic interactions, van der waals interactions, chemical couplings, dielectric or zwitterionic interactions, or a combination thereof.

8. The method of claim 1, wherein the contact medium comprises a compressed gas selected from compressed helium, argon, xenon, nitrogen, carbon dioxide, or a combination thereof.

9. The method according to claim 1, wherein the biological sample is mounted on a substrate, for example a substrate selected from glass, silicon, poly-L-lysine coated materials, nitrocellulose, polystyrene, Cyclic Olefin Copolymer (COC), Cyclic Olefin Polymer (COP), polypropylene, polyethylene, and/or polycarbonate.

10. The method of claim 9, wherein the biological sample comprises a nucleic acid analyte and the contact medium comprises a particulate material comprising silica.

11. The method of claim 1, further comprising microdissection, such as laser microdissection.

12. The method of claim 1, wherein the contact medium is removed from the biological sample by: vacuum processes, pressure differentials or gradients, gravity, transport media (e.g., liquid or aerosol or gas), or transport media selected from magnetic or electric fields.

13. The method of claim 1, comprising: (a) selectively contacting a non-target Region (RONI) in the biological sample with a contact medium, wherein the selective contacting preferably comprises keeping the target Region (ROI) in the biological sample out of contact; (b) selectively contacting a target Region (ROI) in the biological sample with a contact medium, wherein said selective contacting preferably comprises leaving non-target Regions (RONI) in the biological sample untouched; or (c) contacting both the ROI and the RONI in the biological sample with a contact medium.

14. The method of claim 13, comprising: collecting particulate matter in the contact medium; (c) optionally preparing a particulate material for analysis; (d) the particulate matter is further optionally analyzed.

15. The method of claim 14, 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.

16. The method of claim 14, wherein, 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, immuno PCR (ipcr), enzymatic 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, micro-electro-mechanical systems (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), electrochemiluminescence ELISA, and chemiluminescence ELISA.

17. The method of claim 13, further comprising mixing the contact medium with an enrichment medium.

18. The method according to claim 17, wherein 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.

19. The method of claim 18, wherein the biological sample comprises a two-dimensional tissue (e.g., a tissue slice or sheet) or a three-dimensional tissue (e.g., a tissue mass) containing well-defined spatial locations of a region of interest (ROI) and/or a non-region of interest (RONI); well-defined spatial locations containing both ROI and RONI are preferred.

20. A method of determining an analyte in a biological sample, the method comprising processing the biological sample by: (a) contacting the biological sample with a contact medium comprising particulate matter and a compressed gas under conditions sufficient to effect at least partial transfer of components 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 treated biological sample or the removed analyte in the contact medium.

21. A biological analysis system comprising: (a) a first component for contacting the biological sample or a region therein with a contact medium comprising particulate matter and a compressed gas under conditions sufficient to effect at least partial transfer of components in the biological sample to the contact medium; (b) a second component for removing the contact medium from the biological sample; (c) optionally a third component for analyzing components in the contact medium or the treated biological sample or both the contact medium and the treated biological sample.

22. The biological analysis system of claim 21, wherein the first component includes a pressurized Particulate Microjet (PMB) containing a contact medium.

23. The biological analysis system of claim 21, wherein the second component comprises a vacuum, a pressure differential or gradient, a medium for transporting particulate matter (e.g., a liquid or aerosol), or a transmission medium selected from a magnetic or electric field.

24. The biological analysis system of claim 21, wherein the optional third component includes 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 sphere formation, electrophoresis, micro-electro-mechanical 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), electrochemiluminescence ELISA, and chemiluminescence ELISA.

Technical Field

The present disclosure relates generally to the field of histology and/or pathobiology. More particularly, the present disclosure relates to pre-analysis substrate processing systems and related methods, which in some embodiments can be used to separate and isolate a target Region (ROI) and a non-target Region (RONI) in a substrate (e.g., a biological substrate, such as a histological sample).

Background

Various methods have been proposed to address the cumbersome nature of the doctor blade procedure for preparing and/or processing histological samples for downstream analysis. For example, it has been proposed to use vacuum blasting techniques; however, these can only be deployed in an industrial environment, thus requiring expensive tools and instrumentation. In addition, there is currently no vacuum blasting technique compatible with substrates mounted on microscope slides. Similarly, particle-based blasting methods, such as blasting with, for example, silica particles, silica-coated particles, or polymer-coated ferromagnetic beads, require that the particles be separated from the region of interest (ROI) before downstream analysis steps are performed.

The current tissue cutting process is entirely manual, using a razor blade to collect the "S" (i.e., ROI) region directly from the substrate. For example, a conventional workflow for substrate rendering (substrate rendering) includes a completely manual process in which a user manually transfers pathologist target area markers on a stained slide to an unstained slide using a standard, off-the-shelf marker. The user then uses a razor blade or similar instrument to scrape off the target area on the marked, unstained substrate and collects it in a container.

The described manual process limits the operator's accuracy by relying entirely on the operator's hand/eye coordination, which can affect the consistency and accuracy of the tissue blade. This process also introduces ergonomic/safety issues, as the application of constant force to the glass surface can lead to operator nicks and ergonomic issues (e.g., carpal tunnel).

The current implementation of digital pathology has improved many aspects of the histology/pathology workflow. However, there are still some important aspects of unmet needs. One unmet need is that the presentation of certain substrates cannot be digitally processed using commercial digital pathology systems. Manual glass workflow processes are still required in many cases. Therefore, a method is needed to convert the process to achieve a complete digital workflow.

Disclosure of Invention

Some embodiments presented in the present disclosure relate to an automated tissue cutting (ATD) system. The ATD system is a one-station and potentially low-cost system that can be cut on a basement using non-contact and/or mechanical methods from pathologist digital or pen marks on the basement to extract Formalin Fixed Paraffin Embedded (FFPE) tissue samples: (a) only one or more ROIs are taken as regions to be saved; (b) the nucleic acid content in the non-target Region (RONI) is removed or lysed and all tissue samples are collected from a standard microscope substrate into a specific container.

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

In some embodiments, the system is capable of performing the following operations: (a) capturing stained and unstained slide images at appropriate magnification, (b) digitizing the pen markers into digital markers, (c) performing an object-based or other algorithm to match the marker coordinates on the substrate to the relevant substrate, (d) extracting the marked sample ROI into a container, (e) removing the sample in the RONI or resolving nucleic acids in the RONI and collecting all samples (e.g., tissues) into the container, and (f) lysing the sample and outputting the lysate.

This technique may be useful because current manual processes may have certain drawbacks or limitations, such as: (a) the quality is poor; (b) operator limitations; (c) work risk; (d) ergonomics and/or (e) safety. For example, manual cutting methods depend entirely on the hand/eye coordination of the individual operator, which may affect the consistency and accuracy of the results. They may also require specialized training programs to achieve consistent accuracy. Manual cutting with a constant force on the glass surface can also cause ergonomic problems for the operator over a period of time; such as wrist injuries, e.g. carpal tunnel syndrome. Thus, in many laboratories, the operator must also limit the time it takes to make a manual cut to help prevent injury. There is also a risk of damage to the razor blade from blade breakage in terms of safety, for example, the blade may cause nicks. However, current laboratory procedures typically use a completely manual method, for example, manually marking a pathologist's target area using a standard off-the-shelf marker, where the end user can use a razor blade or similar tool (i.e., scalpel) to shave the target area on the substrate.

Sample cutting may be performed using an automated system; however, such systems may be expensive and/or low throughput. For example,MILLISECT^TMthe 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, this system is expensive, slow to operate, and may require many systems and operators for a large volume laboratory. Thus, this system may not be suitable for high throughput sample analysis. Laser capture cutting systems are also available from several manufacturers (e.g., the Leica LMD6-7 system). In these systems, the ROI is cut (e.g., delineated or scribed by a boundary) using a UV laser, and an adhesive cap (adhesive cap) is inflated using an IR laser to remove the ROI from the substrate (e.g., to enhance release of the sample from the substrate); the system also requires a customized substrate that can be collected for subsequent analysis.

The present disclosure addresses several aspects of sample cutting that may cause difficulties, and embodiments may include different levels of system complexity. The present systems and methods provide a method of separating a region of interest (ROI) on a substrate that can provide higher throughput, thereby providing faster sample cutting, while avoiding manual separation and use of standard slide equipment.

The present disclosure includes, for example, a system for processing a sample immobilized on a substrate, the system comprising: (a) a holder unit for fixing a substrate; (b) a camera placed against the cradle unit; (c) a processing element configured to remove a portion of the sample immobilized on the substrate; (d) a computing device communicatively connected to the rack unit.

According to aspects, the camera and processing element may comprise: (a) an image capture engine configured to acquire, using the camera, 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 marking 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 marking image onto the second image to rotate and align the image outlines of the first fixed sample and the second fixed sample, and (d) a sample removal engine configured to control positioning of the holder 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.

Thus, some system embodiments herein include a means for digitally marking that demarcates an interface between one or more ROIs on a sample (also referred to herein as an "S" sample) and other samples on a slide (also referred to herein as an "X" sample). Such interfaces include, for example, algorithms that allow for the creation of virtual markers on samples by virtually tracking manual markers on parallel samples that were previously placed on the same sample or that have been manually aligned with the sample, or automatically aligning previously manually marked parallel samples with a target sample, and virtually tracking markers from one sample to another. Such devices also include the mechanical components necessary to perform these actions.

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 ROIs 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, electrical current, ultrasound, micro-blasting, micro-bead blasting, particle blasting, sand blasting, 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 that can specifically direct a mechanism such as a laser or water jet to the X sample while avoiding ROIs.

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 acid, strong base, or one or more enzymes that selectively direct to the X sample rather than to the ROI on the slide, as well as the mechanical components required to employ these methods and algorithms that can direct chemicals specifically to the X sample while avoiding the ROI.

In some system embodiments, the system further comprises means for pass-through collection of one or more ROIs on the substrate. For example, a pass-through may not include a label because all samples were collected. Once X is killed or removed, a pass-through method may be used, as all remaining samples (e.g., only S-zone) will be collected. Exemplary means include, for example, particle blasting, a blade such as a razor blade, scalpel, spatula, spoon, punch, vacuum source that allows vacuum removal of the sample, or a charged surface or medium that provides a competitive surface or medium or solution (e.g., particle micro-blasting) for the ROI sample, or the use of viscous media to extract the ROI from the slide. Such means also include other mechanical elements that automatically control the use of the above elements as desired.

In some embodiments, the system comprises means for placing the ROI sample into a container. Exemplary means include, for example, mechanical components that can automatically control the following processes: the ROI sample removed from the slide is removed and placed into or onto a container such as a well structure, tube or vial.

Aspects of the present disclosure describe a method for processing a sample immobilized 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 marker image comprising a digital outline of the first image and a portion of the first fixed sample; (d) superimposing the marker image on the second image to align image contours of the first and second fixed samples; and (e) removing only a portion of the second fixed sample within the digital profile of the first fixed sample using a processing element.

Other aspects of the present disclosure describe a system for processing a sample secured to a substrate, the system comprising: (a) a holder unit for fixing a substrate; (b) a camera placed against the cradle unit; (c) a processing element configured to provide a nucleic acid denaturing agent to denature nucleic acids on a portion of the sample immobilized on the substrate; and (d) a computing device communicatively coupled to the cradle unit.

According to aspects, the camera and the processing element may comprise: (a) an image capture engine configured 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 using the camera, (b) a digital marking engine configured to allow a user to generate a marking image comprising the first image and a digital outline of a portion of the first fixed sample, (c) an image overlay engine configured to overlay the marking image on the second image to align the image outlines of the first and second fixed samples, and (d) a nucleic acid denaturation engine configured to control positioning of the scaffold unit and processing elements to denature nucleic acids in only an 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.

A further aspect describes a method for processing a sample immobilized 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 marker image comprising a digital outline of the first image and a portion of the first fixed sample; (d) superimposing the marker image on the second image to align image contours of the first and second fixed samples; (e) denaturing nucleic acids in only a portion of the second fixed sample within the digital profile of the first fixed sample using a processing element.

In another aspect, systems, methods, and devices for processing histological samples, such as FFPE slides or fixed tissue, are disclosed. These systems, methods and apparatus overcome the limitations of existing Particle Microjet (PMB) and Computer Numerical Control (CNC) milling technologies, greatly improving ease of use and sensitivity and/or specificity of the analysis workflow. In particular, the present disclosure relates to variations of PMB and CNC milling techniques that allow for the efficient removal of non-target Regions (RONI) or collection of target Regions (ROI) from histological samples while minimizing loss of analytes (e.g., nucleic acids, proteins, and other macromolecules) in the ROI. The described method is simple and can be seamlessly integrated with downstream analysis programs.

In accordance with the present disclosure, a combination grit blasting technique is used to treat a biological sample mounted on a substrate, such as a slide. A combination of blasting material (i.e., particles) and compressed air is forced into the system and directed to the patient's tissue ROI. The PMB is configured to contain the blasted patient tissue within a wall of the PMB. Next, the blasted tissue is removed from the area using vacuum and accumulated into a container. A filter may be used to separate the container to create a vacuum but prevent portions from reaching the vacuum pump. The method can be used for blasting waste areas ("X") or target areas ("S") and also for blasting all target contents. The blasting media may be made of various materials including, for example, alumina; silicon dioxide; a metal-based particle; magnetic or ferromagnetic particles; salts, ionic particles, lyophilized reagent particles.

In contrast to prior PMB techniques that require separation of particles from the target final target (e.g., prior to downstream processing), the systems and methods of the present disclosure do not require separation of particles from the processed sample because the particles themselves are used for downstream processing.

The method of the invention is also advantageous over prior methods in that it allows multiple processing steps to be integrated into one step, which helps to reduce cross-contamination, and also allows for collection of analytes, such as nucleic acids, from tissue samples in a highly simplified, efficient and inexpensive manner. By reducing operator considerations, the presently disclosed systems and methods help to reduce the variability and/or increase the reproducibility of histological tests (e.g., immunohistochemistry, nuclease protection tests, etc.).

Another advantage of the present technology is that the systems and/or methods of the present disclosure can be modularly applied to existing analytical workflows, such as downstream analytical procedures, e.g., microscopy, hybridization assays, sequencing, chromatography, spectrometry, ELISA, and the like. This means that the presently disclosed systems and/or methods can be seamlessly integrated in various pre-processing and post-processing steps. The system and method may also be applied as a scaffold for various downstream analysis steps, if desired.

The methods of the present invention can be readily modified or adapted to utilize a variety of ablation 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 may be used, such as hydrophobic, polar, non-polar, ion-exchange capable particles, affinity-specific particles, dielectric particles, lyophilized particles, and the like. Moreover, because the reagents and systems used herein are relatively inexpensive, the present techniques can be readily integrated into analytical workflows to significantly improve performance without significantly increasing assay costs.

Accordingly, the present disclosure is directed, in part, to the following non-limiting embodiments:

in some embodiments, the present disclosure relates to a method of processing a biological sample for a bioassay, the method comprising: (a) contacting the biological sample with a contact medium comprising particulate matter and compressed air under conditions sufficient to effect 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.

The present disclosure relates to a method of processing a biological sample for bioassay, for example, for analyzing one or more analytes for diagnostic purposes, according to the foregoing or following embodiments, wherein the biological sample comprises a punch biopsy, a needle biopsy, fresh tissue, a tissue culture, a frozen tissue sample, neutral formalin treated tissue, an organ, an 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.).

The present disclosure relates to a sample milling device comprising first and second components. The first part has openings at both ends. The second member is secured to one end of the first member and has a sample collection port facing away from where the second member is secured to the first member. The sample collection port has one or more sample scraping elements protruding along the perimeter of the sample collection port. A vacuum channel extends through the first and second members to connect the sample collection port with a vacuum connection port on the other end of the first member.

The present disclosure relates to a method of processing a biological sample for biological assays (e.g., for analysis of one or more analytes of diagnostic interest) according to the foregoing or following embodiments, comprising contacting a region of interest (ROI), a non-region of interest (RONI), or all regions in the biological sample with a contact medium; preferably, the target Region (ROI) is brought into contact with the contact medium.

The present disclosure relates to a method of treating 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 at least one analyte of diagnostic interest 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, or viral coat proteins; preferably a nucleic acid selected from mRNA, gDNA, viral DNA or viral RNA.

The present disclosure relates to a method of processing a biological sample for biological assays (e.g., for analysis of one or more analytes of diagnostic interest) according to the preceding or following embodiments, wherein the particulate matter in the blasting media comprises alumina; silicon dioxide; a metal-based particle; magnetic or ferromagnetic particles or combinations thereof.

The present disclosure relates to a method of processing a biological sample for biological assays (e.g., for analysis of one or more analytes of diagnostic interest) according to the preceding or following embodiments, wherein the particulate material is capable of binding to an analyte or a non-analyte in the biological sample; preferably, the particulate matter is capable of binding the analyte via an interaction selected from ionic interactions, polar-non-polar interactions, hydrophobic interactions, van der waals interactions, chemical couplings, dielectric or zwitterionic interactions, or a combination thereof.

The present disclosure relates to a method of processing a biological sample for biological assays (e.g., for analysis of 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.

The present disclosure relates to a method of processing a biological sample for biological assays (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, e.g., a substrate selected from glass, silicon, poly-L-lysine coated materials, nitrocellulose, polystyrene, Cyclic Olefin Copolymer (COC), Cyclic Olefin Polymer (COP), polypropylene, polyethylene, paper, and/or polycarbonate.

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

The present disclosure relates to a method of processing a biological sample for biological assays, e.g., for analysis of one or more analytes of diagnostic interest, further comprising microdissection, e.g., laser microdissection, according to the foregoing or following embodiments.

The present disclosure relates to a method of processing a biological sample for biological assays, e.g., for analysis of one or more analytes of diagnostic interest, according to the preceding or following embodiments, wherein the contact medium is removed from the biological sample by: vacuum processes, pressure differentials or gradients, gravity, transport media (e.g., liquids, aerosols, or gases), or transport media selected from magnetic or electric fields.

The present disclosure relates to a method of processing a biological sample for biological assay, e.g., for analysis of one or more analytes of diagnostic interest, according to the preceding or following embodiments, the method comprising: (a) selectively contacting a non-target Region (RONI) in the biological sample with a contact medium, wherein the selective contacting preferably comprises keeping the target Region (ROI) in the biological sample out of contact; (b) selectively contacting a target Region (ROI) in the biological sample with a contact medium, wherein said selective contacting preferably comprises leaving non-target Regions (RONI) in the biological sample untouched; or (c) contacting both the ROI and the RONI in the biological sample with a contact medium.

The present disclosure relates to a method of processing a biological sample for biological assays, e.g., for analysis of one or more analytes of diagnostic interest, according to the preceding or following embodiments, comprising collecting particulate matter in a contact medium; (c) optionally preparing a particulate material for analysis; (d) the particulate matter is further optionally analyzed.

The present disclosure relates to a method of processing a biological sample for biological assay, e.g., for analysis of one or more analytes for diagnostic purposes, according to the preceding or following embodiments, wherein preparing 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, assays for particulate matter include 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), immunoassays, immuno PCR (ipcr), enzymatic activity assays, 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, micro-electro-mechanical systems (MEMS) electrophoresis, mass spectrometry, chromatography (e.g., HPLC), proximity ligation assays, electrochemical detection, plasmon resonance (SPR), hybridization assays (e.g., in situ hybridization assays, such as Fluorescence In Situ Hybridization (FISH)) FRET, cell sorting (e.g., FACS), electrochemiluminescence ELISA, and chemiluminescence ELISA.

The present disclosure relates to a method of processing a biological sample for biological assay, e.g., for analysis of one or more analytes of diagnostic interest, according to the preceding or following embodiments, wherein the method further comprises mixing the contact medium with an enrichment medium. Preferably, in this embodiment, the enrichment medium comprises a substance that specifically binds to the 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.

The present disclosure relates to a method of processing a biological sample for biological assays, e.g., for analysis of one or more analytes of diagnostic interest, according to the foregoing or following embodiments, wherein the biological sample comprises a two-dimensional tissue (e.g., a tissue slice or sheet) or a three-dimensional tissue (e.g., a tissue mass) comprising a region of interest (ROI) and/or a non-region of interest (RONI); preferably containing both the ROI and RONI well-defined spatial positions.

In some embodiments, the present disclosure relates to a method of 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 a compressed gas under conditions sufficient to effect at least partial transfer of components 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 treated biological sample or the removed analyte in the contact medium.

In some embodiments, the present disclosure relates to a biological analysis system comprising: (a) a first component for contacting the biological sample or a region therein with a contact medium comprising particulate matter and a compressed gas under conditions sufficient to effect at least partial transfer of components in the biological sample to the contact medium; (b) a second component for removing the contact medium from the biological sample; (c) optionally a third component for analyzing components in the contact medium or the treated biological sample or both the contact medium and the treated biological sample.

The present disclosure is directed to a biological analysis system of the foregoing or following embodiments, wherein the first component comprises a pressurized Particle Microjet (PMB) comprising a contact medium.

The present disclosure is directed to a biological analysis system of the foregoing or following embodiments, wherein the second component comprises a vacuum, a pressure differential or gradient, a medium for transporting particulate matter (e.g., a liquid or aerosol), or a transmission medium selected from a magnetic field or an electric field.

The present disclosure relates to a biological analysis system of the foregoing 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 sphere formation, electrophoresis, micro-electro-mechanical 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), electrochemiluminescence ELISA, and chemiluminescence ELISA.

Some embodiments presented in the present disclosure relate to an automated tissue cutting (ATD) system. The ATD system is a one-station and potentially low-cost system that can be cut on a basement using non-contact and/or mechanical methods from pathologist digital or pen markings on the basement to extract formalin-fixed paraffin-embedded (FFPE) tissue samples: (a) only one or more ROIs are taken as regions to be saved; (b) the DNA in the non-target Region (RONI) was removed or lysed and all tissue samples were collected from a standard microscope base into a special container.

Drawings

Fig. 1 shows a schematic of various doctor blade processes used according to various embodiments.

Fig. 2 provides a flow diagram illustrating an exemplary process for digital processing substrate rendering, according to various embodiments.

Fig. 3 provides a flow diagram illustrating an improved method for collecting "S" regions or removing "X" regions, according to various embodiments.

Fig. 4 illustrates an exemplary method for sample cutting according to various embodiments.

Fig. 5 illustrates exemplary transmitted and reflected radiation incident on a mask according to various embodiments. This is because the radiation is partially blocked by the mask and the radiation can only reach certain areas (e.g., only a certain percentage of the energy can be transmitted as a function of wavelength).

Fig. 6 shows an exemplary slide with marked unwanted "X" regions and target Regions (ROIs), according to various embodiments. For example, the "X" region and ROI may be determined by the process described below.

Fig. 7 illustrates a process of removing unwanted "X" regions from a substrate by overlaying a mask on the substrate, according to various embodiments. For example, the mask can be prepared as follows. A mask may be placed over the substrate and the "X" regions may be removed by various methods as described below.

Fig. 8A and 8B show representative illustrations of particle microjets according to various embodiments. Fig. 8A shows a representative implementation of PMBs of the present disclosure for processing patient samples; fig. 8B shows a representative implementation of PMBs of the present disclosure for processing patient samples.

FIG. 9 illustrates an exemplary system architecture for a computer system for implementing the described systems and methods.

Fig. 10 illustrates an exemplary sample processing device according to various embodiments.

Fig. 11 illustrates an exemplary sample processing device according to various embodiments.

Fig. 12 illustrates an exemplary sample processing system according to various embodiments.

Detailed Description

Definition of

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

In this application, the use of "or" means "and/or" unless stated otherwise. In the context of several dependent claims, the use of "or" merely refers to one or more of the preceding independent or dependent claims in the alternative claims.

The word "about" means a range of plus or minus 10% of the value, e.g., "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 interpretation. For example, in a numerical list such as "about 49, about 50, about 55," about 50 "refers to a range that extends less than half of the interval between a previous value and a subsequent value, e.g., greater than 49.5 to less than 52.5. Further, the phrase "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.

Where a range of numerical values is provided in the present disclosure, it is intended that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. For example, if the 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.

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

As used herein, the term "detecting" 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, detection of a tumor includes identifying, determining, measuring, and/or quantifying one or more markers.

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

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

As used herein, the term "sample" refers to a composition obtained or derived from a subject of interest that comprises cells and/or other molecular entities that are characterized and/or identified, e.g., 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 may be blood or any blood component; a body fluid; solid tissue from fresh, frozen and/or preserved organ or tissue samples or biopsies or aspirates; and cells or plasma from the subject at any time during pregnancy or development. 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 tissue, tumor tissue, and cell extracts. Samples also include biological samples that have been treated for certain components (e.g., proteins or nucleic acids) by, for example, reagent treatment, lysis or enrichment, or biological samples embedded in a semi-solid or solid matrix for sectioning purposes, such as tissue or cell thin sections in histological samples. The sample may contain environmental components such as water, soil, mud, air, resins, minerals, and the like. 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).

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 tissues, 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, and the like. Mammalian cells can be, for example, from humans, mice, rats, horses, goats, sheep, cattle, primates, and the like.

As used herein, the term "tumor" includes any cell or tissue that may have undergone transformation at a genetic, cellular, or physiological level as compared to a normal or wild-type cell. The term generally refers to neoplastic growth that may be benign (e.g., tumors that do not form metastases and destroy adjacent normal tissue) or malignant/cancerous (e.g., tumors that invade surrounding tissue and are generally capable of producing metastases, which may recur upon attempted removal, and which are likely to result in host death unless properly treated). See Medical Dictionary,28, Steadman^th Ed Williams&Wilkins,Baltimore,MD(2005)。

The term "cancer" refers to abnormal cell growth, particularly cancers and carcinomas that are malignant in nature, sarcomas, adenocarcinomas, lymphomas, leukemias, solid and lymphoid cancers, and the like. 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 osteogenic sarcoma, 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 bowel cancer, central nervous system cancer, skin cancer, choriocarcinoma, osteogenic sarcoma, fibrosarcoma, glioma, melanoma, and the like.

The term "normal" as used in the context of "normal cells" refers to cells of an untransformed phenotype or cells (e.g., PBMCs) that exhibit the morphology of untransformed cells of the tissue type under examination. In some embodiments, a "normal sample" as used herein includes a non-tumor sample, such as a saliva sample, a skin sample, a hair sample, and the like. It should be noted that the methods of the present disclosure can be implemented without using a normal sample.

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

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 anti-cancer agent). Representative types of markers include, for example, molecular changes in the structure (e.g., sequence) or number of the marker, including, for example, genetic mutations, genetic repeats, amino acid substitutions, additions or deletions, or multiple differences, such as somatic changes in DNA, copy number variations, tandem repeats, translocations, or combinations thereof.

As used herein, the term "genetic marker" refers to a polynucleotide sequence having a particular location on a genome or corresponding to a particular location in a genome (e.g., a transcript complementary to the sequence of a genomic location). Thus, the term "genetic marker" may also be used to refer to, for example, cDNA and/or mRNA encoded by a genomic sequence, as well as the genomic sequence itself. The genetic marker may comprise two or more alleles or variants. Genetic markers include nucleic acid sequences that encode or do not encode a gene product (e.g., a protein). In particular, genetic markers include single nucleotide polymorphisms/variations or copy number variations or combinations thereof. Preferably, the genetic marker comprises a somatic variation in DNA, such as snv or sCNV, an indel, SV or a combination thereof, as compared to the reference sample.

As used herein, the term "protein tag" or "proteomic tag" refers to a sequence of a polypeptide or fragment thereof (e.g., a biologically active fragment of a polypeptide) that corresponds 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).

As used herein, a "formalin-fixed wax-embedded" or "formalin-fixed paraffin-embedded" or "FFPE" tissue sample is to be broadly construed as referring to a sample that has been fixed with formalin or an equivalent material and embedded in wax (e.g., paraffin or an equivalent material). The FFPE tissue herein may be from any human, animal or plant source.

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

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

As used herein, the term "particulate" material refers to a material consisting of particles, such as substantially spherical particles or relatively irregularly shaped particles. Typically, the particulate matter is from about 10nm to about 100 μm in diameter; preferably, about 50 to about 400 nm; particularly about 100 to about 200 nm.

As used herein, the term "assay" is a test for the quantity, presence or absence of a substance.

As used herein, the term "compressed" gas refers to a gas that has been compressed, for example, at a pressure greater than atmospheric pressure. The "gas" component in this 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.

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, or the like, and incubating at a temperature and for a time sufficient to allow interaction between the target and the agent.

In the context of in vivo diagnosis or treatment, "contacting" refers to introducing an active ingredient (e.g., a compound or drug) into a subject and contacting the active ingredient with a target tissue (e.g., epithelial tissue) of the subject in vivo.

As used herein, the term "subject" refers to any animal, preferably a mammal, such as a human, veterinary or farm animal, livestock or pet, including animals commonly used in clinical studies. In particular, the subject is a human subject, e.g., a human patient diagnosed with a disease such as cancer. A subject may have, potentially have or be suspected of having one or more characteristics associated with a disease, one or more symptoms associated with a disease, be asymptomatic or undiagnosed with respect to the disease. In particular, the subject may have cancer, the subject may exhibit symptoms associated with cancer, the subject may not have symptoms associated with cancer, or the subject may not be diagnosed with cancer.

As used herein, the term "noise" refers in the broadest sense to any unwanted interference (e.g., signals that are 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 the system by man-made and natural sources. Noise distorts the signal, degrading the quality or reliability of the information carried by the signal. This term is in contrast to "signal", which is a function that conveys information about the behavior or attributes of a certain phenomenon, such as the probabilistic association between a marker (SNV, CNV, indel, SV) and a disease (e.g., cancer).

As used herein, the term "estimate" is used in a broad sense in the context of marker levels. As such, the term "estimate" may refer to an actual value (e.g., 1 variations per mbp DNA), a range of values, a statistical value (e.g., mean, median, etc.), or other manner of estimation (e.g., probabilistically).

As used herein, the term "substantially" means sufficient for the intended purpose. Thus, the term "substantially" allows for minor, insignificant variations from absolute or perfect states, dimensions, measurements, results, etc., which would be expected by one of ordinary skill in the art, but which would not significantly affect overall performance. "substantially" when used with respect to a numerical value or a parameter or characteristic that may be expressed as a numerical value means within ten percent.

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

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 natural environment (e.g., mitochondria in the case of mitochondrial DNA) to a non-natural environment (e.g., the surface of a silica particle) by processes such as binding, bonding, adsorption, and the like. The term includes processes such as covalent or non-covalent interactions between components and non-natural systems.

As used herein, the term "covalent" interaction involves the sharing of 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 combinations thereof.

As used herein, the term "analyte" generally refers to a target molecule that is detected using the methods or systems disclosed herein. The analyte may 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 may include, for example, a polynucleotide, a polypeptide, a carbohydrate, a lipid, or a combination of one or more of these. Typically, macromolecules have molecular weights of at least about 300 daltons, and can be millions of daltons. Small molecules are organic compounds having a molecular weight of up to about 300 daltons. In some cases, the analyte is a nucleic acid analyte.

As used herein, a "probe" is a substance, e.g., a molecule, that can recognize or be specifically recognized by a particular target. Types of potential probe/target or target/probe binding partners include receptors/ligands; ligand/anti-ligand (anti-ligand); nucleic acid (polynucleotide) interactions, including DNA/DNA, DNA/RNA, PNA (peptide nucleic acid)/nucleic acid; enzymes with substrates, small or effector molecules, other catalysts or other substances, and the like. Examples of probes contemplated by the present invention include, but are not limited to, peptides, enzymes (e.g., 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 may be linear or circular. Probes can distinguish between different targets by differential activity, differential binding, or identification by structural labeling. The probe of the present invention is preferably a nucleic acid molecule, particularly preferably DNA. In some cases, a "probe" may function as a "target" and a "target" may function as a probe, e.g., complementary dna (cdna) may 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.

As used herein, the term "analyzing" and the phrase "detecting" may refer to qualitatively or quantitatively determining a parameter of interest associated with an analyte, such as an amount, level, concentration or activity (absolute and relative) of the analyte.

As used herein, the term "diagnosis" refers to a method by which it is possible to determine whether a subject is likely to suffer from a given disease or disorder. The skilled artisan typically diagnoses based on one or more diagnostic indicators (e.g., markers) whose presence, absence, amount, or change in amount is indicative of the presence, severity, or absence of a disease or disorder. Other diagnostic indicators may include patient history; physical symptoms such as unexplained weight loss, fever, fatigue, pain, or skin abnormalities; a phenotype; the genotype; or environmental or genetic factors. One skilled in the art will appreciate that the term "diagnosis" refers to an increased likelihood of occurrence of a certain process or result; that is, a course or outcome is more likely to occur in a patient exhibiting a given characteristic (e.g., the presence or level of a diagnostic marker) than in an individual not exhibiting that characteristic. The diagnostic methods of the present disclosure can be used alone or in combination with other diagnostic methods to determine whether a patient exhibiting a given characteristic is more likely to develop a course or outcome.

The term "nucleic acid" generally refers to DNA or RNA, whether it is an amplification product, synthetically produced, 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 may have 3 'or 5' overhangs and therefore need not be or are assumed to be completely double-stranded over their entire length. Furthermore, the term nucleic acid may consist 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 ligation or prevent exonuclease degradation/polymerase extension, respectively; covalent and near-covalent attachment of amino, thiol, alkyne or biotin-based modifications; a fluorophore and a quencher; phosphorothioate, methylphosphonate, phosphoramidate and phosphate linkages between nucleotides to prevent degradation; methylated and modified bases.

As used herein, the term "polypeptide" refers to peptides, proteins or polypeptides used interchangeably and encompass amino acid chains of a given length in which the amino acid residues are linked by covalent peptide bonds. The term polypeptide also refers to and does not exclude modifications of the polypeptide. Modifications include glycosylation, acetylation, acylation, phosphorylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, covalent cross-link formation, cysteine formation, pyroglutamate formation, formulation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenization, sulfation, transfer of RNA mediated amino acid addition proteins (e.g., arginylation), and ubiquitination.

As used herein, in the context of molecules, the term "isolated" or "extracted" refers to molecules that are substantially free of impurities. Molecules (e.g., DNA or RNA) have been "isolated" or "extracted" when purified from other components in a sample. Purification refers to the separation of the target from one or more external components also found in the sample. The components isolated, extracted or purified from the pooled sample or samples are 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 the unpurified or unextracted sample.

The term "synthetic" refers to a molecule that has been chemically synthesized using techniques understood in the art, for example, using phosphoramidite chemistry or synthetic chemistry.

In the context of nucleic acids, the term "hybrid" or "hybridization" is broadly intended to include duplexes as well as molecules capable of so forming duplexes. Herein, a single-stranded nucleic acid that base pairs at multiple bases is referred to as "hybridization". Hybridization is typically determined under physiological or biological conditions (e.g., intracellular: pH7.2, 140mM potassium ion; extracellular: pH 7.4, 145mM sodium ion).

As used herein, the term "analog" includes, but is not limited to, oligonucleotides having synthetically introduced residues or linkers therein, 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. Likewise, 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 linked to one or more fluorescent dye molecules.

As used herein, "target" refers to a substance whose presence, activity and/or amount is to be determined and which has an affinity for a given probe. The target may be a man-made or naturally occurring substance. Furthermore, they can be used in the unaltered state or together with other species as agglomerates. The target may be covalently or non-covalently attached to the binding member, either directly or via 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 cleaving neurotransmitters, proteases, kinases, etc.); a substrate for an enzyme; a peptide; a cofactor; a lectin; a sugar; a polysaccharide; cells (which may include cell surface antigens); a cell membrane; organelles, etc., and other such molecules or other substances that may exist in complexed, covalently cross-linked, etc., form. The target may also be referred to as an anti-probe.

Where the probe binds to the target sequence, the binding may be "specific" or "selective". Typically, a probe has the property of being "specific" if it has one and only one binding partner (e.g., target). In fact, the vast majority of probes are "selective" rather than "specific" in that most probes bind many targets, especially at high concentrations. Accordingly, these terms may be used interchangeably. Specificity and selectivity of binding can be determined using conventional methods. For example, where the target is a particular mRNA, the probe may be, for example, an oligonucleotide that specifically binds to the target but not to interfering RNA or DNA under the selected hybridization conditions. One skilled in the art can experimentally determine the characteristics of an oligonucleotide that will optimally hybridize to a target with minimal hybridization to non-specific interfering DNA or RNA using art-recognized methods (e.g., see above). In general, the length of the oligonucleotide probes used to distinguish target mrnas present in a background of large excess of non-targeted RNA can range from about 8 to about 50 nucleotides, preferably about 18, 20, 22, or 25 nucleotides. Oligonucleotide probes used in biochemical assays without a large background of competing targets may be shorter than 8 nucleotides. Using art recognized programs (e.g., the computer program BLAST), the sequences of the oligonucleotide probes can be selected such that they are independent of each other and differ from potentially interfering sequences in known genetic databases. The selection of hybridization conditions that allow specific hybridization of the oligonucleotide probe to the RNA target can be routinely determined using art-recognized procedures.

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 examples is used to prime the synthesis of a longer nucleic acid sequence. The length of the longer primer may be about 10, 12, 15, 20, 25, 30, or 50 nucleotides or longer. Primers can also be used for detection.

Herein, "mechanically removing or ablating" certain samples means mechanically separating the sample from the substrate or vaporizing or decomposing the sample so that it is no longer present on the substrate.

Chemically "breaking down" a macromolecule herein refers to denaturing or breaking down a macromolecule, such as RNA, DNA, and/or protein, or chemically modifying it sufficiently so that it does not contaminate subsequent analysis of RNA, DNA, and/or protein in ROI tissue.

The "marking" made on the slide by a pathologist or laboratory user or other individual is referred to herein as "manual marking". Such markings may be made by any available means, such as with a pen or an etching device. In contrast, a mark that is automatically made by the system herein may be referred to as a "virtual mark" or a "digital mark" to indicate that the mark is not made manually, but is made by using one or more algorithms.

In contrast to operations that are performed manually by a user, the terms "digital," "digitized," "automated," and "automatic" and the like refer to operations performed by the system herein, e.g., controlled by algorithms and/or through user interaction with a computer user interface.

As used herein, the term "surface" refers to any substance that provides a site that allows for interaction between an analyte or target probe. Preferably, the surface is that of a solid support such as nitrocellulose, the walls of the wells of a reaction tray, multiwell plates, test tubes, polystyrene beads, magnetic beads, membranes and microparticles (e.g., latex particles). The term contemplates a suitable porous material having sufficient porosity to allow access by detection reagents and suitable surface affinity to immobilize 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 that have a gel structure in the 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 with nitric acid and carboxylic acid), 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 which may be prepared with suitable porous structures, for example vinyl polymers including polyethylene, polypropylene, polystyrene, polyvinyl chloride, polyvinyl acetate and partially hydrolysed derivatives thereof, polyacrylamides, polymethacrylates, copolymers and terpolymers of the above polycondensates (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 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 polymeric materials described above); and mixtures or copolymers of the above classes, for example graft copolymers obtained by initiating the polymerization of synthetic polymers on existing natural polymers.

As used herein, the term "characteristic" refers to a collection of markers indicative of a phenotype of interest, e.g., a cancer characteristic comprising ≧ 3 mutations which indicates that the cell or tissue carrying the mutation is a tumor cell. In some embodiments, the features include the presence, absence, and/or abundance of a combination of markers, e.g., tumor markers. By combining various probe sets, a reliable method for detecting a target phenotype can be devised. Such feature testing as a single assay may provide great benefit for assessing and understanding the interaction between various markers.

The term "amplification" generally refers to the production of a plurality of nucleic acid molecules from a target nucleic acid, wherein a primer hybridizes to a specific site on the target nucleic acid molecule to provide an initiation site for extension by a polymerase. Amplification can be performed by any method commonly known in the art, such as, but not limited to, standard PCR, long PCR, hot start PCR, qPCR, RT-PCR, and real-time PCR.

The term "antibody" as used herein refers to an intact immunoglobulin, such as IgA, IgD, IgE, IgG or IgM, or a fragment (particularly an antigen binding fragment) of an antibody, 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 radioisotope.

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

The term "fixed tissue or cell" is used herein and refers to a biological tissue or cell that is preserved from decay by chemical fixation methods, as known to those skilled in the art. Such methods may prevent autolysis or spoilage within such biological tissues or cells. Fixation can terminate biochemical reactions and increase the mechanical stability of the treated tissue.

The term "immunohistochemistry" or "IHC" refers to a technique for detecting the presence of an antigen in a histological sample with an antibody capable of specifically binding to said antigen. The detection of the antibody-antigen complex is usually performed by a color reaction using an enzyme-labeled antibody or by a fluorescent-labeled antibody.

As used herein, the term "macro-dissection" refers to the process of scraping a target area from a section of tissue mounted on a solid support such as a microscope slide by using a tool such as a cutting knife or scraper. As used herein, the term "microdissection" refers to the process of cutting and isolating one or more specific cells or target regions from a tissue sample. Microdissection can be performed, for example, using Laser Capture Microdissection (LCM) by cutting the relevant area with laser light.

As used herein, the term "membrane slide" refers to a solid support or microscope slide for Laser Capture Microdissection (LCM). For microdissection, slides covered with a film or framed slides consisting of a metal frame covered with various films can be used.

The term "polylysine" refers to a molecule comprising up to several hundred repeat 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. Polylysine according to the description is poly-L-lysine. The poly-L-lysine according to the description has a molecular weight of 70 to 300 kDa. poly-L-lysine can be digested by proteases. Another polylysine according to the description is, for example, poly-D-lysine. The poly-D-lysine according to the description has a molecular weight of 70 to 300 kDa. poly-D-lysine is resistant to protease digestion.

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 target nucleic acids, where quantification is by means of an embedded fluorescent dye or sequence specific probe comprising a fluorescent reporter molecule that is only detectable after hybridization to the target nucleic acid.

The term "RNA" is used herein and refers to mRNA precursors, mRNA precursor transcripts, mrnas, transcript processing intermediates, mature mrnas for translation and nucleic acids derived from one or more gene transcripts, or derivatives thereof, as known to those skilled in the art. Transcript processing includes processes such as splicing, editing, modification and degradation. The mRNA comprising the sample includes, for example, 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.

A "container" that may include a sample herein is to be broadly construed to mean any type, shape, or size of container, including a surface, well, tube, or vial. The container need not have any particular shape or size made of any particular material, but rather serves merely as a physical structure capable of analyzing or manipulating tissue located therein or thereon.

A "stained" slide/substrate herein refers to a substrate that has been processed to help reveal differences between ROI samples 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 treatment.

The term "isolating" one or more ROIs from an X sample on a substrate includes methods of processing the X sample in such a way that: the X sample is physically removed from the substrate, ablated (e.g., vaporized or burned or physically decomposed), or chemically treated, and thus does not contaminate subsequent analysis of the molecules in the ROI sample.

The term "substrate" herein refers to a variety of slides including, but not limited to, FFPE slides, tissue slides, standard slides, containers, stained slides, unstained slides, biopsy, and the like.

The term "sample" herein refers to a cellular tissue, a specimen, a tissue sample, FFPE tissue or other biological material that is fixed using standard molecular biology methods.

Herein, a "computer processor" or "computing device" or "computer" is to be broadly interpreted to mean any combination of hardware and/or software that will perform its intended function. For example, the processor may 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). Where the processor is programmable, appropriate programming may be communicated to the processor from a user interface incorporated in the computer body or located at a remote location, or may be pre-stored in a computer program product (e.g., a portable or fixed computer-readable storage medium, which may be magnetic, optical, or solid-state based). For example, a magnetic medium or optical disk may carry the programming and may be read by a suitable reader in communication with each processor at its corresponding user interface. "user interface" is broadly construed herein to mean a physical structure that allows a user to program a computer and thereby control certain operations of a system via the computer. Examples include keyboards and monitors for a host or portable computer, other types of visual monitors and keyboard systems, such as tablet or smartphone-type devices or other remote devices. The user interface may be a physical part of the computer body, may be located elsewhere in the system, may be located remotely from the computer, and may communicate with the computer processor through a wired or wireless connection.

Method for digitally marking ROIs on undyed substrates

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

Macroscopic cutting techniques involving not only histological sections of the target region but also histological sections of the surrounding tissue of the organ to be investigated have been increasingly used in many pathological studies, such as tumor typing, diagnosis of inflammatory diseases and determination of degenerative diseases. In macroscopic dissection, target patient tissue ("S" region) is collected from a tissue sample (e.g., slide) while excluding unwanted regions ("X" regions), thus using only the "S" region as input material for downstream assays. In some cases, the complex "S" shape definition provided by the pathologist may make scraping operations difficult for the tissue technician. Often requiring sophisticated skiving techniques, as shown in fig. 1. The present disclosure relates to systems and methods for processing biological samples so that analytes therein can be more accurately detected, 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 bioassays, particularly in the context of analyzing analytes in heterogeneous samples (e.g., FFPE).

The presently disclosed systems and methods also improve assay objectives by improving assay parameters, such as signal quality and reducing noise, for example, correlating the presence/absence or level or activity of a biomarker with a target feature, such as an ethnic feature (for forensics) or a disease feature.

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

The presently disclosed method may be used in a variety of settings. For example, in a first implementation in which macroscopic cutting is desired, the "X" regions in the substrate (e.g., a histological slide) can be selectively micro-blasted, leaving only the "S" regions on the substrate unaffected. Thereafter, downstream processing methods may be used to remove the "S" regions from the substrate. In a second implementation, where macroscopic cutting is also desired, the "S" regions in the substrate (e.g., a histological slide) can be selectively micro-blasted, leaving only the "X" regions on the substrate unaffected. The tissue of the "S" region can be removed from the substrate and the particles collected in a container for processing using downstream processing. In another implementation, where macro-cutting is not required, all regions of the substrate ("X" and "S" regions) are micro-blasted and the contents of both regions are collected in a vessel for downstream processing.

Fig. 2 shows an exemplary process for digitally processing unstained slide presentation (USS), where quality and tissue thickness cannot be uniformly controlled. The process may include two parallel processes with three paths that may result in a parallel process merge.

According to one aspect, the USS may be mounted to a bracket along a first parallel path. The installed USS may be stored in a storage unit to wait for a path check. A scanned image of the USS is created. The USS is retrieved from the storage unit. An automatic sample cutting procedure may then be performed.

According to one aspect, the USS is stained along a second parallel path to produce a reference slide. The reference slide is scanned to create an image. A digitized mark is created that includes a region of interest (ROI) on the reference slide. It is then determined whether the flag can be uploaded to an Image Management System (IMS). If so, the second parallel path merges with the first parallel path through path 1.

In pathway 1, the presentation substrate successfully transferred its label through current digital pathology solutions. The USS can be processed earliest in the current digital workflow through path 1.

If not, an external label transfer algorithm is applied. If the label can be transferred, path 2 connects the second parallel path to the first parallel path. For example, in path 2, rendering the substrate requires development of label transfer algorithms external to the current digital pathology solution. This path assumption requires an external label transfer algorithm (e.g., capabilities in current digital pathology solutions).

Finally, the label can be transferred manually through path 3. For example, in path 3, label transfer cannot be performed directly from the presentation substrate, and a pathologist is required to perform digital labeling manually on each relevant substrate. In the case where neither path 1 nor path 2 can continue execution, this path is a backup solution. The process can still be handled at least in an automated sample scraping system.

FIG. 3 illustrates a method of collecting "S" regions or removing "X" regions. According to one aspect, the "X" region is removed and only the "S" region is left to allow a straight-through (all samples collected from the slide) method for sample collection. This is an effective method because there is typically only a small "X" area. For example, about 50% of cases are straight-through (e.g., no "S" region). In most cases, the "X" regions are much smaller than the "S" regions (e.g., fewer "X" regions to remove).

Other aspects include breaking down the nucleic acid in the "X" region (e.g., leaving only the "S" region and the inactive "X" region) so that the "X" region need not be removed 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. Thus, there will be no analyzable amount of DNA or RNA in the "X" region.

Additional aspects include lysing the sample directly on the substrate as a straight-through method. This method can partially break down the phospholipid bilayer of the cells and/or completely break down the proteins, thereby processing the sample into a liquid lysate suitable for downstream analytical processes. For example, the sample may be exposed to a lysis buffer or buffer solution. Examples of lysing solutions include: hypertonic, hypotonic, pH adjusted solutions or solutions containing enzymes (e.g. proteases).

The described process may be applicable to both automated and manual workflows. For example, they can be implemented in conjunction with current manual scraping techniques because there are many more "S" regions than "X" regions, and thus "X" regions are easier to remove or decompose than if only "S" regions were collected.

Sample cutting process

Fig. 4 illustrates various sample cutting methods. According to aspects of the present disclosure, removing the "X" region may leave only the "S" region on the slide for collection. For example, all unwanted samples on the substrate can be removed, leaving only the wanted sample on the slide for straight-through extraction. To remove the "X" regions, mechanical and non-mechanical tools can be used to extract unwanted samples from the substrate.

Mechanical (e.g., contact) tools may include the use of physical materials such as razor blades, machine milling (standard or custom milling tools), curettes, punches, spoons, bead blasting, and vacuum suction to remove unwanted samples directly from the substrate. The machine tool may also include a particle blasting process for removing the "X" regions.

Non-mechanical means 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, pulse laser systems, Q-switch laser systems, Nd: YAG laser system, continuous laser system, dye laser system, tunable laser system, titanium sapphire laser system, high power diode laser system, continuous wave laser or high power fiber laser system. For example, a femtosecond laser system may utilize an average power of 20W, which has a size of a mid-tower (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 appreciated that the laser utilized may be configured to be lower power to produce fewer traces (focprintings) than conventional lasers.

Nucleic acids (e.g., DNA) may be decomposed (e.g., denatured) in the "X" region, leaving only the "S" region and inactive "X" region on the slide for collection. For example, the breakdown of nucleic acids in the "X" regions will render those regions inactive for subsequent processes (e.g., the nucleic acid content is not at a level that affects gene expression). Thus, there is no need to physically remove/cut those areas, and a pass-through sample collection method can be used.

Non-mechanical methods, such as laser, thermal, RF, ultrasonic, cryogenic, plasma, etc., can be used to decompose unwanted "X" regions from a substrate (e.g., a slide).

Chemical methods may include applying chemical agents (e.g., bleaches, acids, bases, enzymes, etc.) to the "X" region to break down nucleic acids (e.g., DNA/RNA). NaOH or salt may also be used to denature nucleic acids. Additional denaturants may include protein denaturants and nucleic acid denaturants. An exemplary concentration of bleaching agent may be at least 10% bleaching agent. Combinations of chemicals may be combined, including pH adjusted solutions containing endonucleases and/or proteases or 0.05% -10.0% (weight/volume) sodium hypochlorite.

According to various aspects, performing the lysis process directly on the substrate using a lysis buffer may bypass the sample cleavage process. For example, the substrate with the sample can be directly immersed in a temperature-controlled container with lysis buffer to separate the entire sample from the substrate. Subsequent protein kinase (ProK) protein digestions can be performed in the same vessel and in separate processes/vessels.

According to aspects of the present disclosure, sample cutting may include mechanical and non-mechanical methods to extract unwanted samples (e.g., unwanted "X" regions) from a substrate. As described above, mechanical (contact) methods may include the use of physical materials to cut unwanted samples directly from a substrate. The system may utilize a physical scraping tool (e.g., a particulate micro-blast, a sand blast, a razor blade, a curette, a punch or a spoon) in conjunction with a vacuum suction tube after the scraping action to simultaneously collect unwanted samples using a suction method.

The aspiration device consists of a low cost disposable consumable (e.g., plastic tube with a filter) to collect all samples and will be replaced in each case to avoid cross-contamination. Alternatively, the aspiration device may be cleaned between samples to eliminate cross-contamination. The filter has the function of stopping the flow of the sample but allowing the passage of air. In addition, the sample cannot completely block the filter and thus block the passage of air.

The withdrawn sample may then be collected in a waste bin, leaving only the "S" region on the substrate in the straight-through method. For example, cut-through collection may be achieved by: (a) the automatic direct single-pass razor blade direct connection can be easily realized; (b) separating the sample from the substrate into a container/tube using a temperature controlled ultrasonic water bath; and/or (c) applying an electrostatic charge to the bottom of the collection vessel/tube after the sample is separated from the substrate.

Non-mechanical methods may include the use of indirect techniques (e.g., such as laser and water jet ablation) to separate unwanted samples from the substrate. For example, laser and water jet methods may include applying a force on a target glass surface using a high pressure water jet or laser abrasion technique to separate a sample from a substrate. For the through method, this results in leaving only "S" regions on the substrate.

Straight-through collection can be accomplished by: (a) the automatic direct single-pass razor blade direct connection can be easily realized; (b) separating the sample from the substrate into a container/tube using a temperature controlled ultrasonic water bath; and/or (c) applying an electrostatic charge to the bottom of the collection vessel/tube after the sample is separated from the substrate.

To decompose the "X" region, the following method can be used to extract unwanted samples from the substrate. For example, non-mechanical methods can break down the DNA in the unwanted "X" region from the substrate. According to one embodiment, the system will use a non-contact ablation technique, such as laser, thermal, RF, ultrasound, cryogenic, or plasma, to decompose the unwanted sample from the substrate. For the through-pass approach outlined above, this results in leaving only "S" regions on the substrate.

The chemical method may include applying a chemical to the "X" region to break down the DNA. For example, the system may apply a chemical agent (e.g., bleach, acid, alkali, or enzyme, etc.) on a target "X" region of the glass surface to break down the sample from the substrate. For the through-pass approach outlined above, this results in leaving only "S" regions on the substrate.

It should be understood that the present disclosure is not limited to the field of pathology. It can also be used for pre-enrichment or isolation of targets as well as non-targets. Certain techniques may also be used alone or in combination with other integrated systems. It will also be appreciated that the method for straight-through may also include the use of pneumatics or a combination of the above methods.

Sample types include, but are not limited to, cell cultures, frozen sections, fresh samples, liquid biopsies, and cytological samples (i.e., sputum, pleural fluid, etc.). The sample type may also include non-human targets.

The target sample is also not limited to the substrate. Any form of container may be provided as an input to the system to enable the system to image the sample. Other examples include coverslips (i.e. to produce a blood smear), bioreactors, cell culture dishes with imaging punches, sample collection papers or streams/droplets.

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

In some embodiments, the cutting system can use many means such as laser, water jet, and ultrasound to cut the substrate to separate the ROI from the unwanted sample.

Some embodiments may include a low cost mechanical system that utilizes milling techniques. A small bench CNC milling machine uses a vertical end mill/spoon (scooper) to collect predefined numerical labels of ROI (samples to be preserved) or RONI (samples to be discarded) samples. The desktop system allows a user to handle a set of slides in a milling housing, and a vertical milling tool (e.g., a low cost rotary scraping fixture with suction features) will be able to track and collect samples with predefined final user digital annotations. In some embodiments, the preserved sample may 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 shaved with a razor blade.

For example, a large fraction (e.g., more than 40%) of biopsy samples from certain cancers may be all S without X, while many other samples have relatively small X regions. If a small end mill arrangement is used, the end mill may have a lumen for aspiration (i.e., the grinder may be a hypotube (hypotube) with or without a grinding tip to simplify machining of the sample). If the X area is small, the X area can be removed. If the S region is small, the S region can be machined and collected. For simplicity, it is also possible to remove all X regions or collect all S regions in unison. In some embodiments, the X sample on the slide can alternatively be removed using water jet, leaving behind the S sample.

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 micro-blasting (e.g., bead blasting), laser, electrolysis, ultrasound, radio frequency, or thermal energy sources) or by selectively freeze-drying the X sample. In some embodiments, the means necessary to ablate the X sample comprises an energy source, such as a laser, radio frequency, electrical current, sound, or thermal energy source capable of lysing cells and/or breaking down biological macromolecules (e.g., nucleic acids and proteins). For example, some of these methods can effectively burn and evaporate X samples. In some embodiments, the device can precisely direct a suitable energy source to an area on the substrate other than the pen or digital marker, for example, precisely focusing a laser or radio frequency or ultrasound wave 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 break down nucleic acids in the X region of the slide, such that only the S region containing the ROI remains on the slide. For example, the system may be configured to direct only a pulsed laser, electrolytic or ultrasonic device to the X region once tracking of the S and X regions has been performed. For example, the slide may be scanned from end to end only with energy from a laser, electrolysis device, or ultrasound device, contacting only the X region of the slide, thus 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 be effective to evaporate the sample in the X regions and can destroy target molecules, such as DNA and RNA, in those regions so that the resulting isolated S regions are not contaminated by material from the X regions in subsequent analyses.

In other embodiments, the X-region can be effectively removed by chemical treatment, including aqueous cavitation methods (e.g., ultrasonic aqueous cavitation methods), such as with one or more reagents that break down molecules (e.g., DNA and/or RNA and/or proteins) from the sample to be analyzed. Chemical means for selectively decomposing the X sample include, for example, the addition of bleaching agents, strong acids, strong bases, or enzymes that target and decompose macromolecules (e.g., dnases, rnases, and proteases). For example, chemical treatment with RNase or protease can be directed only to the X region of the slide based on digitized pen marks on the slide. RNases or proteases can, for example, break down RNA or proteins into small fragments or monomers. As a result of the chemical treatment, for example, the X region of the slide will not contain significant levels of intact molecules for analysis, and thus, for example, analysis of the expression level of a protein or RNA in the treated sample will reflect the expression level in the S portion of the sample only, as only the protein and/or RNA from the S portion will remain intact.

Either of these two methods-ablating the 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 S tissue by excising the S tissue from a slide containing the X and S tissues. Alternatively, in a "straight through" tissue collection method, the entire tissue on the slide can be collected or used for later analysis. For example, in a "straight through" collection, all tissue on the slide is removed and there is no tissue cutting between the S and X regions. Thus, the methods herein are compatible with straight-through tissue collection, where separation of the S and X tissues on a slide is not required when collecting the tissues for later analysis. Alternatively, the tissue may simply be removed from the slide, for example by scraping it off the slide and into a collection container by capillary action, suction, or the like with a rotating cutting tool similar to a milling process. In such embodiments, even if tissue collection is performed manually, the highly skilled razor blade technology currently used is not required, which can cause injury or result in low accuracy. Alternatively, tissue collection may be performed automatically by the instrument. This automation is easier to implement because the goal is to collect all tissue without having to separate the X-region from the S-region. For example, the tissue may 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 proteins). Means for mechanically collecting tissue from the treated slide into the receptacle include, for example, sandblasting, scraping the tissue using a razor blade or similar blade, or a curette, spoon, punch or vacuum sucking the tissue, adding a solution or another substance to provide a competitive medium or surface, such as a charged surface or medium, as compared to the slide surface.

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, for example, can be performed directly on the slide. 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 can be performed directly on the tissue slide, e.g., using an appropriate kit, and optionally also extracting target molecules such as DNA, RNA, or proteins. In some embodiments, such a process may be automatically controlled by the device. For example, in some embodiments, the slide can be immersed in a lysis buffer, e.g., contained in a well or tube or vial, so that cell lysis can be performed on the slide. In some embodiments, subsequent steps, such as protease digestion or DNase digestion and/or RNA extraction, may then be performed on the submerged slide.

FIG. 5 is a diagram 500 of an exemplary transmitted illumination 506 and reflected illumination 504 of incident radiation 502 on a mask 508. This is because the radiation 502 is partially blocked by the mask 508 and the transmitted radiation 506 can only reach certain areas of the sample 512 (e.g., only a certain percentage of the energy can be transmitted as a function of wavelength). Substrate 510 may be 1mm thick, which provides some refraction to transmitted illumination 506. For example, the mask 508 may be on the top side of the slide 510 such that the radiation 502 is partially blocked by the mask 508 and the radiation can only reach the "X" area. In accordance with aspects of the present disclosure, the mask 508 may include an optical, thermal, mechanical structure, and/or chemical mask to block applied associated heat, laser, chemical agents (e.g., bead blasting), and the like. It should be understood that the exposed portions may be removed or denatured depending on the process employed.

Fig. 6 shows an exemplary slide 600 having marked unwanted "X" regions 602 and target Regions (ROIs) 604 (e.g., "S" regions) between the "X" regions 602. For example, the "X" region 602 and ROI 604 may be determined by the process described above.

FIG. 7 shows the process of removing unwanted "X" regions 712 and 714 from slide 720 by overlaying mask 700 on slide 720. For example, a mask slide 710 can be prepared having delineated unwanted "X" regions 702, 704 and wanted "S" regions 706. For example, "X" regions 702, 704 and "S" region 706 may be determined by the process described above. Overlaying the mask 700 on the slide 720 allows unwanted "X" regions 712 and 714 to be removed from the slide 720 according to the depicted portions. The resulting slide 720 includes only the desired portion 716. It is understood that "X" regions 712 and 714 may be removed or denatured depending on the process employed.

An exemplary embodiment of an automated tissue cutting (ATD) system utilizes a mini milling system (fig. 12) with a disposable custom designed milling tool as shown in fig. 10 and 11, which depict the distal cutting portion of the 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 rotation of the milling tool. One or more lumens (i.e., channels) may be present in the tool to facilitate the pressing of the severed tissue into the lumens by vacuum suction. The cutting portion may be engaged with the body of the tool. The body functions to receive collected tissue, engage with a machine chuck, receive a filter element and engage with a cutting portion. Compressed or other inert gas may be used to force the severed tissue into the collection tube. Alternative embodiments include gravity transfer into the collection tube under agitation, wash buffer, or placing the milling tool with the collected tissue directly into the test tube. In each case consisting of one or more tissue slides, one disposable milling tool may be used.

To reduce machining time, the custom milling cutter is designed to operate at high rotational speeds with direct drive or pneumatics. The rotational speed may reach or exceed 100,000 rpm. This can increase the feed rate; the tool is therefore designed to withstand the operating stresses and temperatures that occur when operating at high rotational speeds.

The distal cutting region may have a lumen for collecting the tissue ROI and have one or more cutting edges radially arranged on the front face. The material may be steel or any material that is harder than the tissue and facilitates low cost, high volume machining. Engineering plastics may be a good choice because they can be economically injection molded. Since the ROI may be close to 750 microns or less for certain tissue types, the diameter of the front face may be very small. Small lumens can severely increase the pressure differential across the cutting section and restrict airflow. Preferably, the lumen size is rapidly reduced and immediately increased at a location near the cutting interface.

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

The filter element will prevent the passage of collected tissue but allow the passage of air or inert gas. The filter opening size may be 50 microns or greater. The opening size and the overall size of the filter are designed so that the collected tissue does not completely clog the filter at the end of the treatment. The filter needs to withstand operating pressures and forces. A pressure relief feature may also be incorporated to prevent clogging. The filter may be bonded or mechanically attached to the body. Alternative clamping members may also be used.

The distal cutting portion of the milling tool is connected to the body. The distal cutting portion may include one or more cutting edges that engage the tissue slide. The distal cutting section is designed to direct the cut tissue toward the center of the cutting section. One or more cutting edges at the distal position are radially oriented. Each cutting edge may be straight or curved. The contact angle of the blade with the ROI during operation is such that a net vector force will be generated to push tissue towards the center of the treatment element, allowing the ROI to be collected. For example, cutting edge #1 may be set to be vertical, cutting edge #2 may form an acute angle, and cutting edge #3 may be set to be obtuse, each of these positions being set to drive the cut tissue. A preferred embodiment is to provide a distal cutting portion consisting of one or more edges disposed at least as with cutting edge #1, and preferably oriented to have an acute angle as with cutting edge # 2. The distal cutting portion may be designed to enhance cutting capabilities, including variations in serration edges and serration locations, including length, width and depth.

Fig. 10 and 11 illustrate an exemplary sample milling device according to various embodiments. As shown herein, the apparatus 1000 includes a first component 1014 and a second component 1010. The first assembly 1014 has openings on opposite ends, and the second assembly 1010 is secured to one end of the first assembly 1014. The second assembly 1010 further includes a sample collection port 1018, the sample collection port 1018 facing away from where the second assembly 1010 is secured to the first assembly 1014, the sample collection port 1018 having one or more sample scraping elements 1020 protruding along a perimeter of the sample collection port 1018. A vacuum channel 1012 extends through the first and second assemblies 1014, 1010 to connect the sample collection port 1018 with a vacuum connection port 1022 on the other end of the first assembly 1014. When the device 1000 is operated to collect a sample from a substrate, the device 1000 is rotated such that the sample scraping element 1020 mechanically removes a portion of the sample from the substrate surface, while the removed sample portion is collected through the sample collection port 1018 and the vacuum channel 1012 by applying a vacuum to the vacuum connection port 1022 of the first component 1014 using a vacuum pump. In various embodiments, the removed sample portion can be collected by a filter element 1016 connected to the vacuum connection port 1022. In various embodiments, the removed sample portion is collected in a container in fluid communication with the vacuum channel 1012.

In various embodiments, first component 1014 and second component 1010 are comprised of different materials. In various embodiments, the first component 1014 and the second component 101 are comprised of the same material. Examples of materials that may be used include, but are not limited to: metals, polymers, fiberglass, and the like.

In various embodiments, first component 1014 and second component 1010 are fabricated as a single integrated component, rather than as two separate, separate components.

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

Fig. 12 illustrates an exemplary sample collection system according to various embodiments. As depicted herein, the system 1200 includes a sample milling device 1000 located above a substrate (i.e., slide) 1202 that holds a sample 1204. The sample milling device 1000 includes a first assembly 1014 and a second assembly 1010. When the sample milling device 1000 is operated, it is rotated and positioned (in the X, Y and Z axes) such 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 the removed portion of the sample is collected by using a vacuum pump 1206 that applies a vacuum to an opening 1208 on one end of the milling device 1000.

In various embodiments, the sample milling device 100 can be moved in the X, Y, and Z axis directions such that it contacts only a 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-axis directions to cause the sample to mill the device 1000.

In various embodiments, first component 1014 and second component 1010 are comprised of different materials. In various embodiments, the first component 1014 and the second component 101 are comprised of the same material. Examples of materials that may be used include, but are not limited to: metals, polymers, fiberglass, and the like.

Exemplary subsequent sample analysis procedure

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, followed by one or more other treatments, as described above. In some embodiments, DNA and/or RNA and/or proteins, cofactors, membrane lipids, etc. may be extracted from ROI tissue directly or after cell lysis, or may be evaluated in situ.

In some embodiments, the systems and methods herein relate to analysis of DNA in ROI tissue. For example, the systems and methods herein may be used in conjunction with ROI tissue analysis in the following manner: copy Number Variation (CNV), Single Nucleotide Polymorphism (SNP), detection of a point mutation in a particular gene, detection of a deletion or insertion mutation in a gene, detection of transposition, translocation, presence of foreign DNA (e.g., viral or bacterial DNA), methylation of DNA, and the like. In some embodiments, the systems and methods herein can be used in conjunction with analysis of RNA species in ROI tissue, e.g., to determine the level of a particular RNA transcript of a gene or to detect particular alternatively spliced RNA transcripts and their relative levels, 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 particular protein in ROI tissue can be assessed by methods such as immunoprecipitation, ELISA, Western blot, nucleic acids, and the like, such as in situ or after a cell lysis procedure. In some embodiments, ROI tissue may be evaluated to detect the presence or levels of other molecules (e.g., biological cofactors, cell membrane lipids or other components, etc.).

Exemplary systems and methods for analyzing biological samples, such as FFPE tissue mounted on a slide, are shown in fig. 8A and 8B.

Fig. 8A illustrates an exemplary method for processing and/or analyzing a patient sample (e.g., a tumor biopsy) according to the methods described above. The biological sample is subjected to a micro-blasting treatment with a contact medium comprising particles and air. In some embodiments, the contact medium may comprise particles well suited for isolating a particular analyte of interest present in a patient sample. For example, silica particles or silica-coated particles, such as silica-coated ferromagnetic beads, are well suited for isolating nucleic acid markers, such as mrnas with missense mutations or loss-of-function mutations. The particle microjets are loaded with such particles and the nozzles of the microjets are directed at the desired tissue region. For example, in the case of a pathological sample, the desired region may be a region containing nucleic acids, such as cell nuclei that have been stained with an appropriate stain. Alternatively, where the patient sample contains different tissue types, the desired region may be a tissue type or layer containing target cells, for example, in the case of pancreatic cancer, epithelial cells lining the pancreatic duct. The target area is micro-blasted with a contact medium (containing compressed gas and particles) to remove target cells, which are then collected and combined with a tissue lysis buffer to destroy the cells. This results in a cell lysate solution in which the analyte of interest (e.g., in the case of pancreatic cancer, mutant nucleic acids encoding KRAS, TP53, CDKN2A, SMAD4, BRCA1 and/or BRCA 2; see Cicenas et al, cancer (Basel), 28, 9(5), 2017) is adsorbed onto the particles. To increase the adsorption rate, the pH of the buffer solution may be adjusted to be at or below the pKa of the surface silanol groups in the silica particles and the salt content of the buffer is increased. The particles are then washed with a solution to adsorb the analyte on their surface while other components of the lysate solution, such as non-analytes, e.g., proteins and lipids, are washed away. The analyte can be directly analyzed using downstream analytical techniques such as PCR. Alternatively, the nucleic acid adsorbed on the surface of the silica particles is eluted with a suitable eluent before downstream analysis by a nucleic acid detection technique such as PCR. Elution is facilitated by the use of a buffer of low ionic strength and low pH. If desired, the sample may be pretreated using microdissection techniques (e.g., laser microdissection) prior to micro-blasting to further refine and/or target the region of interest (ROI).

Fig. 8B illustrates an exemplary method for processing and/or analyzing a biological sample (e.g., a fixed entomology sample for a museum archive or a contact slide containing soil microorganisms) according to the methods described above. The biological sample is subjected to micro-blasting with a contact medium containing particles and a gas. In some embodiments, the contact medium may comprise particles well suited for isolating a particular analyte of interest present in a patient sample. For example, aluminum particles that have been functionalized with amino, carboxyl, sulfonate, and phosphate groups can be used to isolate specific polypeptide markers. The particle microjets are loaded with such particles and the nozzles of the microjets are directed at the desired tissue region. For example, in the case of a fixed insect specimen, the desired region may be a region containing the target marker, such as the abdomen. The target area is micro-blasted with a contact medium (containing compressed gas and particles) to remove target cells, which are then collected and combined with a tissue lysis buffer to destroy the cells. This results in 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 adsorption rate, the particles may be derivatized depending on the physicochemical properties of the target (e.g., hydrophilic in the case of soluble proteins; hydrophobic in the case of membrane proteins). The particles are then washed with the solution such that the analyte is adsorbed on their surface, while other components of the lysate solution, e.g., non-analytes, such as lipids, are washed away. The analyte can be directly analyzed using downstream analysis techniques such as mass spectrometry. Alternatively, the polypeptides adsorbed on the surface of the aluminium particles are eluted with a suitable eluent before downstream analysis by peptide detection techniques such as immunoblotting or mass spectrometry. If desired, the sample may be pretreated using microdissection techniques (e.g., laser microdissection) prior to micro-blasting to further refine and/or target the ROI.

Computer-implemented system

FIG. 9 is a block diagram that illustrates a computer system 400 upon which an embodiment of the present teachings may be implemented. In various embodiments of the present teachings, computer system 400 may include a bus 402 or other communication mechanism for communicating information, and a processor 404 coupled with bus 402 for processing information. In various embodiments, computer system 400 may also include a memory, which may be a Random Access Memory (RAM)406 or other dynamic storage device, coupled to bus 402 to determine instructions for execution by processor 404. The memory may also be used for storing temporary variables or other intermediate information during execution of instructions to be executed by processor 404. In various embodiments, computer system 400 may also include a Read Only Memory (ROM)408 or other static storage device coupled to bus 402 for storing static information and instructions for processor 404. A storage device 410, such as a magnetic disk or optical disk, may be provided and coupled to bus 402 for storing information and instructions.

In various embodiments, computer system 400 may be coupled via bus 402 to a display 412, such as a Cathode Ray Tube (CRT) or Liquid Crystal Display (LCD), for displaying information to a computer user. An input device 414, including alphanumeric and other keys, may be coupled to bus 402 for communicating information and command selections to processor 404. Another type of user input device is cursor control 416, such as a mouse, a trackball, or cursor direction keys for communicating direction information and command selections to processor 404 and for controlling cursor movement on 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), that allows the device to specify positions 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.

Consistent with certain embodiments of the present teachings, computer system 400 may provide results in response to processor 404 executing one or more sequences of one or more instructions contained in memory 406. Such instructions may be read into memory 406 from another computer-readable medium or computer-readable storage medium, such as storage device 410. Execution of the sequences of instructions contained in memory 406 may cause processor 404 to perform processes described herein. Alternatively, hardwired circuitry may be used in place of or in combination with software instructions to implement the present teachings. Thus, implementations of the present teachings are not limited to any specific combination of hardware circuitry and software.

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

Common forms of computer-readable media include, for example, a floppy disk, a flexible disk, hard disk, magnetic tape, or any other magnetic medium, a CD-ROM, any other optical medium, punch cards, paper tape, any other physical medium with patterns of holes, a RAM, a PROM, and EPROM, a FLASH-EPROM, any other memory chip or cartridge, or any other tangible medium from which a computer can read.

In addition to computer-readable media, instructions or data may also be provided as signals on transmission media included in a communication device or system to provide a sequence of one or more instructions to processor 404 of computer system 400 for execution. For example, a communication device may include a transceiver having signals indicative of instructions and data. The instructions and data are configured to cause one or more processors to implement the functions outlined in the disclosure herein. 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, and the like.

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

The methods described herein may be implemented by various means depending on the application. For example, the methods may be implemented in hardware, firmware, software, or any combination thereof. For a hardware implementation, the processing units may be implemented within 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, micro-controllers, microprocessors, electronic devices, other electronic units designed to perform the functions described herein, or a combination thereof.

In various embodiments, the methods of the present teachings may be implemented as firmware and/or as software programs and applications written in conventional programming languages, such as C, C + +, Python, and the like. If implemented as firmware and/or software, the described embodiments may be implemented on a non-transitory computer-readable medium storing a program for causing a computer to perform the above-described methods. It should be understood that the various engines described herein can be provided on a computer system, such as computer system 400 of FIG. 9, whereby processor 404 will perform the analysis and determinations provided by these engines in accordance with instructions provided by any one or combination of storage component 406/408/410 and user input provided through input device 414.

While the present teachings are described in conjunction with various embodiments, there is no intent to limit the present teachings to such embodiments. On the contrary, the present teachings encompass various alternatives, modifications, and equivalents, as will be appreciated by those skilled in the art.

Further, in describing various embodiments, the specification may have presented the method and/or process as a particular sequence of steps. However, to the extent that the method or process does not rely on the particular order of steps set forth herein, the method or process should not be limited to the particular sequence of steps described. As one of ordinary skill in the art will appreciate, other sequences of steps are possible. Therefore, the particular order of the steps set forth in the specification should not be construed as limitations on the claims. In addition, the claims directed to the method and/or process should not be limited to the performance of their steps in the order written, and one skilled in the art can readily appreciate that the sequences may be varied and still remain within the spirit and scope of the various embodiments.

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

It should also be appreciated that the embodiments described herein may employ various computer-implemented operations involving data stored in computer systems. The operations are those requiring physical manipulations of physical quantities. Usually, though not necessarily, these quantities take the form of electrical or magnetic signals capable of being stored, transferred, combined, compared, and otherwise manipulated. Further, the operations performed are often referred to by terms such as producing, identifying, determining, or comparing.

Any operations that form part of the embodiments described herein are useful machine operations. Embodiments described herein also relate to an apparatus or device for performing these operations. The systems and methods described herein may be specially constructed for the required purposes, 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 with computer programs written in accordance with the teachings herein, or it may be more convenient to construct a more specialized apparatus to perform the required operations.

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 which can thereafter be read by a computer system. Examples of computer readable media include hard disk drives, Network Attached Storage (NAS), read-only memory, random-access memory, CD-ROMs, CD-R, CD-RWs, magnetic tape, and other optical, flash, and non-optical data storage devices. The computer readable medium can also be distributed over network coupled computer systems so that the computer readable code is stored and executed in a distributed fashion.

Examples

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

Example 1: processing a biological sample to obtain a nucleic acid analyte

A histological sample containing the target nucleic acid was treated according to the method described above. The contact medium here comprises particles which are very suitable for nucleic acid isolation. These particles include silica particles or silica-coated particles, such as silica-coated ferromagnetic beads. Particulate microjets are loaded with silica particles to provide an efficient method of isolating nucleic acids from tissue samples. First, the desired tissue area is removed from the slide by microspraying with silica particles, which are then collected and combined with tissue lysis buffer to destroy the cells. This produced a cell lysate solution in which the nucleic acids were adsorbed onto silica particles. The silica particles are then washed with a solution that adsorbs the nucleic acids on their surface while washing away other components of the lysate solution, such as proteins and lipids. Nucleic acids can be analyzed directly using downstream analysis techniques such as PCR. Alternatively, the nucleic acid adsorbed on the surface of the silica particles is eluted with a suitable eluent before downstream analysis by a nucleic acid detection technique such as PCR. The sample may be pretreated using microdissection techniques (e.g., laser microdissection), if desired.

Example 2: processing biological samples to obtain nucleic acid and peptide analytes

Alternatively, the particles used for the micro-blasting process in example 1 above are collected in one container and combined with other particles most suitable for selective binding of various potential analytes (e.g. nucleic acids or proteins), and the resulting mixture of particles is used to separate the analytes of interest. As in example 1, in this alternative arrangement, the microprojection particles are first selected to bind (e.g., based on charge or hydrophobicity or affinity) to the target analyte. The resulting mixture of tissue and particles resulting from the microspray is combined with a lysis buffer to disrupt the cells, and the resulting lysate solution is then combined with particles having oligonucleotides or antibodies bound to their surfaces, wherein the oligonucleotides or antibodies specifically bind the target analyte. The particle pairs are then analyzed directly (e.g., chromatographically or spectroscopically). Alternatively, these particle pairs are subjected to a series of binding, washing and elution steps to elute the analyte of interest, which is then analysed using conventional techniques. Finally, where the target analyte is a protein, the lysate solution may also be subjected to other analytical procedures, such as enzymatic assays, binding assays, functional assays, and the like.

The above methods may be combined with any separation and/or purification step, which may be performed at any stage of the workflow, preferably before a final analysis step such as PCR (in the case of nucleic acid analytes) or ELISA (in the case of protein analytes). For example, target nucleic acids are typically isolated from tissue by a series of steps including: (a) tissue lysis, in which the cells of the tissue are disrupted by various methods to rupture open the cells and release their contents into solution; (b) adsorbing the nucleic acid to a solid surface; (c) washing the solid phase with a solution to adsorb the nucleic acids to the solid phase, but to remove other biomolecules; and (d) washing the solid phase with a solution to elute the nucleic acid from the solid phase such that the collected eluate comprises the isolated nucleic acid. In this method, a silica membrane and silica particles are generally used as a solid phase. Other types of particles are also commonly used, including silica-coated or polymer-coated ferromagnetic beads.

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.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this 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, the materials, methods, and examples are illustrative only and not intended to be limiting. In case of conflict, the present specification, including definitions, will control.

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

Description of selected embodiments

Embodiment 1. a system for processing a sample immobilized on a substrate, comprising: a holder unit for fixing a substrate; a camera positioned adjacent to the cradle unit; a processing element configured to remove a portion of the sample immobilized on the substrate; and a computing device communicatively connected to the cradle unit, the camera, and the processing element, including an image capture engine configured 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 using the camera; a digital marking engine configured to allow a user to generate a marked image comprising a digital outline of the first image and a portion of the first fixed sample; an image overlay engine configured to overlay the marker image onto the second image to align image contours of the first and second fixed specimens; and a sample removal engine configured to control the positions of the holder unit and the processing element to remove only a portion of the second fixed sample that is located in the digital profile of the first fixed sample.

Embodiment 2. the system of embodiment 1, wherein the processing element is configured to remove a portion of the sample with a mechanical tool.

Embodiment 3. the system of embodiment 2, wherein the mechanical tool comprises at least one of a grit blast, a razor blade, a milling tool, a curette, a punch, a scoop, a vacuum.

Embodiment 4. the system of any of embodiments 1-3, wherein the processing element is configured to remove the portion of the sample with a non-mechanical tool.

Embodiment 5 the system of embodiment 4, wherein the non-mechanical tool comprises at least one of a laser or a water jet.

Embodiment 6. a method for processing a sample immobilized 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 marker image comprising a first image and a digital outline of a portion of a first fixed sample; superimposing the marker image on the second image to align image contours of the first and second fixed samples; removing only a portion of the second fixed sample that is located in the digital profile of the first fixed sample using a processing element.

Embodiment 7 the method of embodiment 6, wherein the removing comprises removing the portion of the second fixed sample with a mechanical tool.

Embodiment 8. the method of embodiment 6 or embodiment 7, further comprising: removing the portion of the second fixed sample with at least one of sand blasting, razor blade, curette, punch, scoop, vacuum, or combinations thereof.

Embodiment 9 the method of embodiment 6, wherein the removing comprises removing the portion of the second fixed sample with a non-mechanical tool.

Embodiment 10 the method of embodiment 6 or embodiment 9, further comprising removing the portion of the second fixed sample with at least one of a laser or a water jet.

Embodiment 11. a system for processing a sample immobilized on a substrate, comprising: a holder unit for fixing a substrate; a camera positioned adjacent to the cradle unit; a processing element configured to provide a nucleic acid denaturing agent to denature nucleic acids on a portion of the sample immobilized on the substrate; and a computing device communicatively connected to the cradle unit, the camera, and the processing element, including an image capture engine configured 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 using the camera; a digital marking engine configured to allow a user to generate a marked image comprising a digital outline of the first image and a portion of the first fixed sample; an image overlay engine configured to overlay the marker image onto the second image to align image contours of the first and second fixed specimens; and a nucleic acid denaturation engine configured to control the positions of the rack unit and the processing element so as to denature nucleic acid in only a portion of the second fixed sample located in the digital profile 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 cellular analysis.

Embodiment 12 the system of embodiment 11, wherein the nucleic acid denaturing agent comprises a chemical agent.

Embodiment 13 the system of embodiment 12, wherein the chemical agent comprises at least one of a bleaching agent, an acid, a base, or an enzyme.

Embodiment 14 the system of any of embodiments 11-13, wherein the processing element is configured to remove the portion of the sample with a non-chemical tool.

Embodiment 15 the system of embodiment 14, wherein the non-chemical tool comprises at least one of a laser, a thermal heater, a Radio Frequency (RF) wave, ultrasound, cryogenics, or plasma.

Embodiment 16: a method for processing a sample immobilized 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 marker image comprising a digital outline of the first image and a portion of the first fixed sample; superimposing the marker image on the second image to align image contours of the first and second fixed samples; denaturing, using a processing element, nucleic acids in only a portion of the second fixed sample that is located in the digital profile of the first fixed sample.

Embodiment 17 the method of embodiment 16, wherein said denaturing comprises exposing said portion of said second fixed sample to a chemical reagent.

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 bleaching agent, an acid, a base, or an enzyme.

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

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, a Radio Frequency (RF) wave, ultrasound, cryogenic temperature, or plasma.

Embodiment 21: a sample processing device, comprising: a first component having openings on opposite ends; a second component secured to an end of the first component, wherein the second component further comprises a sample collection port facing away from where the second component is secured to the first component, the sample collection port having one or more sample scraping elements protruding along a perimeter 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.

Embodiment 22 the sample processing device of embodiment 21, wherein the first component and the second component are comprised of different materials.

Embodiment 23 the sample processing device of embodiment 22, wherein the first component and the second component are comprised of the same material.

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.

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

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.

Embodiment 27 the sample processing device of embodiment 21, wherein the first component and the second component are fabricated as a single integrated device.