WO2025188332A1 - Histology protection barrier - Google Patents

Abstract

Methods, techniques, and kits are provided herein for purifying cells using molecular targeting, at a cellular or subcellular level. The techniques comprise labeling a biological sample comprising cells with a probe capable of directing the deposition of a hydrophobic protective barrier. The hydrophobic protective barrier is deposited onto the surface of the labeled cells or structures, to protect and retain the biological material under the barrier. A micropurification solution is applied to the biological sample, wherein the micropurification solution dissolves, degrades, digests, or otherwise processes cells not covered by the barrier, allowing for; 1) isolation and collection of the target cells for molecular analysis, 2) isolation and collection of the non-target cells for molecular analysis, 3) removal for the purpose of disposing of the non-target cells. In some aspects, a plurality of probes, each specific to a different target, may be used. The techniques may be performed without the need for complex instrumentation involving microscopy.

Description

HISTOLOGY PROTECTION BARRIER

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63466757, filed May 16th, 2023, the contents of which are hereby incorporated by reference in their entireties.

FIELD OF THE INVENTION

[0002] Histology methods and systems are provided that target and allow isolation of specific types of cells or structures using novel high-precision processing techniques.

BACKGROUND

[0003] Biological samples such as histological tissue sections are inherently complex, containing numerous cell types (e.g., nerve cells, vasculature cells, epithelium cells, fibroblasts, etc.) as well as extracellular structures (e.g., extracellular matrix, etc.) that enable their specialized physiological functions. Quantitative molecular analysis of cells from histological slides is useful in developing a deeper understanding of fundamental pathophysiology and in generating effective clinical interventions [1-15], While this complexity enables specialized functions of each tissue type, it can confound scientific or clinical analysis of individual types of cells within the tissue microenvironment (TME).

[0004] For pathological diseases such as cancer, isolating tumor cells is further complicated by tumor heterogeneity, as a histology sample may comprise normal cells and cancerous cells, as well as regions of hyperplasia or neoplasia. Additionally, a tumor focus may contain multiple clones arising during tumor development and progression, with each clone having a unique expression or mutation profde. Thus, a histological tissue section may include a heterogeneous population of normal cells, a heterogeneous population of tumor cells and other structures present within the tissue and/or tumor microenvironment that complicate analysis.

[0005] In the early 1990s, Shibata demonstrated that cells coated with ink by a mechanical process were protected from UV radiation (see, Shibata et al., Am. Joum. of Path. (1992) vol. 141, no. 3, pp. 539-543). However, this technique was not cell-specific, typically covering groups of cells and protecting any cell covered by ink regardless of its particular cell type. [0006] Several other strategies were developed in the 1990s and early 2000s to isolate cells from heterogeneous tissue sections, including manual scraping, micromanipulators, laser capture microdissection (LCM), and mesodissection [16-32], Manual scraping involves removal of cells (e.g., using a razor, scraper, or other suitable tool) in a specific region of the tissue slide, and is typically performed under a microscope. While this technique is low cost, spatial resolution is low and non-target cell types may be captured along with the target cells. Micromanipulators have also been used to manually remove cells, as part of a process that also is typically performed under a microscope.

[0007] Microdissection techniques have also been used to isolate regions of cells from a tissue or histology slide. Such techniques may utilize immunohistochemistry (IHC)-based methods to identify target cells in order to achieve improved yield and precision. For example, immuno-laser capture microdissection has been used as a guide to the dissection process (see, Emmert-Buck et al., Science (1996), vol. 274: 8, pp. 998-1001). Other immunodissection methods also have been developed, including computer-based stain recognition software programs, expression microlabeling (xML), expression microdissection (xMD) (see, Hanson et al., Nature Protocols (2011) vol. 6: 4, pp. 457-467), AutoScanXT Software, etc. While such laser microdissection methods maintain spatial resolution suitable for isolating cells in heterogeneous environments, such techniques remain costly, are labor and time intensive, are performed manually typically under a microscope, and are subject to operator error. Mesodissection platforms offer yet another platform to isolate cells, by utilizing microfluidics tools to extract cells from particular regions of formalin-fixed paraffin embedded tissue. However, mesodissection also is typically performed manually and prone to operator error and is subject to contamination.

[0008] Laser-based dissection technology has greatly advanced modem biology and molecular pathology [8, 18, 33-46], However, in spite of these successes, laser dissection instruments do not offer an easy way to isolate target cells and fail to meet the needs of modem molecular pathology and personalized medicine. Moreover, laser dissection devices are expensive to purchase and maintain, are time- and labor-intensive to use, and are incapable of efficiently dissecting samples at a cellular, or subcellular level of precision.

[0009] With the advent of personalized medicine and in view of the heterogeneity of different types of tumors, techniques to accurately isolate specific types of tumor cells from individual patients for further analysis and with minimal human intervention are needed. Additionally, inexpensive options are needed for investigators or clinicians who lack access to commercial dissection instruments, and such options should allow procurement of cells by cell type in a precise manner.

[00010] Accordingly, while the aforementioned techniques are used, or have been used, for microdissection, they are subject to a variety of drawbacks, are not precise, and are generally not optimized for high throughput techniques involving personalized medicine. As the fields of biological research, molecular pathology, and precision medicine continue to evolve and coalesce, novel techniques will be needed to isolate and study the molecular content of specific cell populations in order to better understand biology and disease processes.

SUMMARY

[00011] Methods, techniques, kits, and systems for isolating and collecting cells at a cellular/subcellular level of precision are provided herein. A biological sample comprising cells is impacted with a protective material that is deposited onto specified cells, subcellular structures, and/or tissue structures, forming a physical and/or molecular barrier that selectively covers the specified cells, subcellular structures, and/or tissue structures, and does not cover nonspecified cells, subcellular structures, and/or tissue structures.

[00012] A micropurification reagent or process, such as a liquid solution, is then applied to the biological sample that selectively degrades or differentially processes cells, subcellular structures, and/or tissue structures not covered by the protective barrier. The labeled cells covered with the barrier are not degraded or processed. After the micropurification process is complete, both the nontarget cells/structures and the target cells/structures may be separately recovered. The nontarget cells are solubilized or otherwise differentially processed by micropurification and can be recovered. The target cells/structures remain on the slide due to the protective effect of the barrier and can be subsequently recovered by slide scraping, further chemical processing steps that includes removal of the protective solution/barrier, or other recovery means. [00013] Specific cells or structures can be recovered from the biological sample (e.g., from the histological slide) because the protective barrier infuses with, surrounds, and/or covers the specific cells, subcellular structures, and/or tissue structures based upon physical, colorimetric, or molecular characteristics (e.g., cell surface antigens, nucleotide sequences, etc.). These characteristics allow target cells to be specifically identified, as for example when labeled by a probe (e.g., an antibody, a nucleotide-based hybridization probe, an affimer, an aptamer, etc.), histological stain, artificial intelligence, computer software, that produces a signal and/or discriminating information useful for directing deposition of the protective barrier on specific cells, subcellular structures, and/or tissue structures for protection from a micropurification solution that degrades or otherwise processes unprotected cells. The high precision of the signal leads to a corresponding high precision in the location of the protective barrier, and this precision allows isolation of a desired cell population with high resolution compared to prior methods. Moreover, the method is not dependent upon a human operator to manually select target cells of interest.

[00014] It is to be understood that this Summary is not intended to identify key or essential features of embodiments of the present disclosure, nor is it intended to be used to limit the scope of the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

[00015] FIG. 1 is a descriptive diagram of an example (example 1) of how this technology may be performed to establish a protective histological barrier to; 1) protect, isolate, and collect a single cell type from complex histological tissue and/or 2) to isolate and collect a single cell type or multiple cell types that are not protected by the protective histological barrier. Step 1 : Target cells of interest in a standard tissue section placed on a microscope slide are identified via immunohistochemistry using a primary antibody that specifically binds only the target cells of interest. Step 2: A secondary antibody, which specifically binds to the primary antibody, is used in the IHC reaction that is conjugated to the molecule biotin. Step 3 : In order to establish the protective barrier, streptavidin-conjugated paraffin that tightly and specifically binds the biotinylated secondary antibody is incubated on the slide to locate the paraffin at the exact cells that bind the primary and secondary antibodies. Step 4: After washing off the unbound conjugated paraffin, the slide is then heated to a temperature above the melting point of the paraffin so that the paraffin infuses and encompasses the target cells. The slide is then cooled to a temperature below the melting point of paraffin to establish a solid, hydrophobic, histological protective barrier for the target cells.

[00016] FIG. 2 is a descriptive diagram of an example (example 2) of how this technology may be performed to establish a protective histological barrier to; 1) protect, isolate, and collect a single cell type from complex histological tissue and/or 2) to isolate and collect a single cell type or multiple cell types that are not protected by the protective histological barrier. Step 1 : Target cells of interest in a standard tissue section placed on a microscope slide are identified via immunohistochemistry using a primary antibody that specifically binds only the target cells of interest. Step 2: A secondary antibody, which specifically binds to the primary antibody, is used in the IHC reaction that is directly conjugated to paraffin, thus targeting the paraffin precisely to where the secondary antibody binds. Step 3: After washing off the unbound secondary antibody- conjugated paraffin, the slide is then heated to a temperature above the melting point of the paraffin so that the paraffin infuses and encompasses the target cells. The slide is then cooled to a temperature below the melting point of paraffin to establish a solid, hydrophobic, histological protective barrier for the target cells.

[00017] FIG. 3 is a descriptive diagram of an example (example 3) of how this technology may be performed to establish a protective histological barrier to; 1) protect, isolate, and collect a single cell type from complex histological tissue and/or 2) to isolate and collect a single cell type or multiple cell types that are not protected by the protective histological barrier. Step 1 : Target cells of interest in a standard tissue section placed on a microscope slide are identified via immunohistochemistry using a primary antibody that is directly conjugated to paraffin and that specifically binds only the target cells of interest. Step 2: After washing off the unbound primary antibody-conjugated paraffin, the slide is then heated to a temperature above the melting point of the paraffin so that the paraffin infuses and encompasses the target cells. The slide is then cooled to a temperature below the melting point of paraffin to establish a solid, hydrophobic, histological protective barrier for the target cells.

[00018] FIG. 4 is a descriptive diagram of an example (example 4) of how this technology may be performed to establish a protective histological barrier to; 1) protect, isolate, and collect a single cell type from complex histological tissue and/or 2) to isolate and collect a single cell type or multiple cell types that are not protected by the protective histological barrier. Step 1 : Target cells of interest in a standard tissue section placed on a microscope slide are identified via immunohistochemistry using a primary antibody that is coated with paraffin and that specifically binds only the target cells of interest. Step 2: After washing off the unbound primary antibody- coated paraffin, the slide is then heated to a temperature above the melting point of the paraffin so that the paraffin infuses and encompasses the target cells. The slide is then cooled to a temperature below the melting point of paraffin to establish a solid, hydrophobic, histological protective barrier for the target cells.

[00019] FIG. 5 is a diagram showing the process of collecting cells that are NOT protected by the solid, hydrophobic, histological protective barrier. A micropurification solution is placed on the slide whereby the slide is incubated for a period of time and at a temperature to remove, digest, and dissolve the unprotected cells into the micropurification solution. The dissolved, unprotected cells can either be discarded and not analyzed, or molecules such as nucleic acids and proteins within the collected unprotected cells can be analyzed using a number of different technologies including nucleic acid sequencing and mass spectrometry proteomics.

[00020] FIG. 6 is a diagram showing the process of collecting cells that ARE protected by the solid, hydrophobic, histological protective barrier. The solid, hydrophobic, histological protective barrier that remains on the target cells after removal of the unprotected, non-target cells is removed and washed away by standard dewaxing methods using reagents such as xylenes and alcohols. Once the solid, hydrophobic, histological protective barrier is removed exposing the target cells, the micropurification solution is placed on the slide whereby the slide is incubated for a period of time and at a temperature to remove, digest, and dissolve the now unprotected cells into the micropurification solution. The dissolved, unprotected target cells can be analyzed using a number of different technologies including nucleic acid sequencing and mass spectrometry proteomics.

[00021] FIG. 7 shows how the hydrophobic protective barrier made by deposition of paraffin/wax using one of the described methods (FIGS. 1-4) protects cells from being dissolved, solubilized, and/or removed from the histological slide by the micropurification solution, and how this approach is used to isolate both protected cells and non-protected cells from the same slide. This figure demonstrates that the hydrophobic protective barrier protects cells so that nonprotected cells can be dissolved, solubilized, and removed for molecular analysis, while also demonstrating that the protective barrier can subsequently be removed to also dissolve, solubilize, and remove previously protected target cells from the slide for separate molecular analysis (FIGS. 5-6). (A) Approximately lul of melted paraffin/wax (heated to 65°C) was placed on one spot of a standard tissue section on a slide that was heated at 65°C. The melted paraffin/wax was allowed to infuse into, around, and on top of the cells for 2 minutes at 65°C followed by removing the slide from 65°C and placed at room temperature for more than 5 minutes to allow the melted paraffin/wax to solidify into, around, and on top of the target cells. (B) The slide was then placed on a heating plate at 50°C (below the melting point of the hydrophobic protective barrier consisting of paraffin/wax) at which point 500ul of micropurification solution was placed on the slide to cover over the entire area of the tissue including over the area protected by the hydrophobic protective paraffin/wax barrier. The slide was incubated at 50°C for 10 minutes to dissolve, solubilize, and remove non-protected cells and cellular structures by the micropurification solution while leaving the protected cells. The dissolved, solubilized, and isolated non-target cells were removed and placed into a tube for preparing nucleic acids for molecular analysis. (C) The same slide that now contains only the target cells protected by the hydrophobic protective barrier was briefly washed in water to remove residual micropurification solution and then processed to remove the hydrophobic protective barrier using standard dewaxing methods using reagents such as xylenes and graded alcohols resulting in exposing the target cells. Once exposed, the target cells were dissolved, solubilized, and removed by placing the slide on a heating plate at 50°C (below the melting temperature of the hydrophobic protective barrier consisting of paraffin/wax) followed by placing 500ul of micropurification solution to cover over the entire area of the tissue including over the area previously protected by the hydrophobic protective paraffin/wax barrier. The slide was incubated at 50°C for 10 minutes to dissolve, solubilize, and remove the previously protected target cells. The dissolved, solubilized, and isolated previously protected target cells were removed and placed into a tube for preparing nucleic acids for molecular analysis.

DETAILED DESCRIPTION

[00022] Histology Micro Protection (HMP) methods and systems are provided that allow high resolution selection of cells (e g., tumor cells) or subcellular structures at a molecular level using a probe that produces a chemical material that covers the cell to which the probe is attached. HMP utilizes and integrates novel probe-based targeting and microprotection effects to isolate specific types of cells or structures in a complex biological sample comprising a plurality of different cell types. The isolated cells can be subsequently analyzed by any of a variety of techniques, including, e.g., expression profiling, Next Generation Sequencing (NGS), etc.

[00023] HMP methods provide increased resolution, higher throughput, and greater ease of use compared to current cell selection methods and technologies such as manual dissection, laser capture microdissection, laser-based tissue microdissection, or an immuno-based dissection method. For example, HMP can recover specific cells or subcellular structures from histology slides within minutes without relying on complex instrumentation involving laser dissection, slide irradiation, and/or microscopy. Using HMP, cells or subcellular structures are recovered based on their molecular characteristics. Accordingly, HMP reagents may be provided as part of a kit to provide cell selection capability in the absence of complex technologies. Additionally, HMP provides a high level of precision, allowing isolation of a specific type of cell with high yield, as compared to current techniques which typically rely on complex instrumentation and produce lower yields than HMP. These techniques may also be used to isolate a specific type of cell present at a low concentration within a biological sample.

[00024] In one aspect, HMP may be used to isolate cells on a histological slide carrying a biological sample. The histological slide may be generated according to techniques known in the art. For example, a biological sample may be sectioned, formalin-fixed, and embedded in paraffin, and may undergo additional processing to expose the antigens (e.g., antigen retrieval) so that probes can bind to the antigen. The slide may then be incubated with the probe such as a primary antibody, wherein the probe attaches to cells of interest based upon the cell's molecular characteristics. A moiety on the probe forms, e.g., directly or as a byproduct, a protective barrier consisting of a protective material, as for example wax/paraffin, that coats the cells to which the probe is attached. The labeled cells may then be incubated in a micropurification solution for a period of time sufficient to solubilize, differentially process, and/or degrade cells that are not coated with the protective barrier. The time required to solubilize, differentially process, and/or degrade cells may vary depending on the tissue but may be, for example, about 10 minutes. The attached labeled cells may then be collected by removal of the protective barrier (such as by standard dewaxing methods) followed by solubilizing, differentially processing, and or degrading the now-exposed, previously-protected cells and placed into another container. This process is shown by example in FIGs. 1-7.

Histology, Cytology, and Cell Culture Slides, Flasks, and Petri Dishes

[00025] Histology samples typically are thinly sliced, or sectioned, such that the individual sections may be stacked (vertically) to reconstruct the 3 -dimensional structure of the biological sample. Accordingly, in some aspects, the histological slide may include extracellular targets, targets on the surface of a cell, or targets within the cell (e.g., the cytoplasm, the nucleus, etc.) that are accessible by the probes.

[00026] In some aspects, the cells are not processed according to histological techniques, but are adhered to other surfaces, including but not limited to, plastic, glass, polymeric surfaces such as gels, etc. Cells may be in a variety of states (not limited to formalin-fixed and/or paraffin-embedded), including frozen sections, live cells, cultured cells, cells subjected to suspension culture, etc.

[00027] Paraffin-embedded tissues on slides may be dewaxed using xylenes, as referenced in the experimental protocols provided herein. Any suitable organic solvent may be used, including, but not limited to xylene(s) benzene, toluene, biphenyl, and the like.

Further, the tissue slide may be placed in the organic solvent from 1 minute up to 30 minutes, or any range in between, or more. After dewaxing the sample can be rehydrated as necessary, using methods that are well-known in the art, including using solution containing decreasing concentrations of ethanol or another water-miscible solvent. Any suitable buffer may be utilized (e.g., Tris, etc ), and the protocol is not intended to be limited to Citra Plus buffer.

Anti en Retrieval

[00028] Formalin-fixed tissues sometimes require an antigen retrieval step before a probe, such as a primary antibody, can specifically bind to cells in the tissues. Cross-linking during fixation can mask antigenic sites and antigen retrieval methods can reverse, at least partially, this cross-linking and expose antigenic sites, allowing probes to bind. Methods of antigen retrieval are well known in the art, and the specific protocols provided herein are not intended to be limiting of the methods that can be used. For example, the slides may be microwaved at maximal power (Sanyo, 1200 Watts) until boiling, wherein boiling may occur for any suitable amount of time, e.g., from 1 second up to 10 minutes or more, or any range in between. The slides may then be microwaved for an additional amount of time at medium power, wherein the amount of time may range from 1 second up to 20 minutes, or more, or any range in between. The slides may be cooled for a period of time ranging from 1 second up to 30 minutes, or more, or any range in between, followed by rinsing with diH2O.

[00029] A single heating step, or multiple heating steps may be used. Any combination or duration of heating steps, e.g., time, temperature, cycles, may be used provided that antigens are exposed and can be bound by a probe, such as a primary antibody, and that damage to the underlying biological material of the target cells is minimized.

Antigens

[00030] A variety of antigens may be used with the HMP methods as described herein. In general, the HMP method may be applied to any biological target, e.g., including but not limited to an extracellular target, a target on the surface of the cell, an intracellular target, a lipid, a protein, a cell type, a subcellular compartment, or any target that may be specifically labeled. A suitable antigen may be specific to a particular type of cell and/or may indicate a cancerous state of the cell (e.g., indicate high proliferation). For example, antigens associated with the presence of cancer include, but are not limited to: alpha fetoprotein (AFP), CAI 5-3, CA27-29, CA19-9, CA-125, calcitonin, calretinin, carcinoembryonic antigen (CEA), CD34, CD99MIC 2, CD117, chromogranin, chromosomes 3, 7, 17, and 9p21, cytokeratin (e.g., TP A, TPS, Cyfra21-1), desmin, epithelial membrane antigen (EMA), factor VIII, CD31 FL1; glial fibrillary acidic protein (GFAP), gross cystic disease fluid protein (GCDFP-15), HMB-45, human chorionic gonadotropin, immunoglobulin, inhibin, keratin, lymphocyte marker, MART-1 (Melan-A), myo DI, muscle-specific actin (MSA), neuron-specific enolase (NSE), placental alkaline phosphatase (PLAP), prostate-specific antigen (PSA), PTPRC (CD45), SI 00 protein, smooth muscle actin (SMA), synaptophysin, thymidine kinase, thyroglobulin (Tg), thyroid transcription factor-1 (TTF-1), tumor M2, and vimentin. [00031] A single antigen may be used with the HMP methods provided herein, for example, an antigen unique to a specific type of immune cell. Alternatively, cocktails of antigens may be used, allowing isolation of multiple types of cells (e.g., the immune repertoire using CD 68, CD4, CD11, etc.). In some aspects, a cocktail of probes may be used to recover multiple types of lymphocytes, as part of a diagnostic workflow to ascertain immunological activation status in a patient’s cancer. As another example, the cocktail may comprise a mix of antibodies that bind to a cocktail of antigens including but not limited to CD6, CD8, CD20, and CD68. The methods and techniques provided herein are not intended to be limited to a particular antibody or combination of antibodies.

Probes - Antibodies

[00032] In some embodiments, the probe may comprise an antibody that is specific for a particular antigen associated with a particular type of cell. In some aspects, the probe is conjugated or complexed with a reactive moiety, such as an enzyme or other inhibitor of a chemical process, or other reporter molecule capable of reacting with a substrate to produce a paraffin/wax material that is deposited on the cells to which the probe is bound. In general, the probe may comprise one or more antibodies, wherein at least one antibody is conjugated to a moiety capable of reacting with a paraffin/wax substrate to produce a hydrophobic protective barrier. In some embodiments, under varying conditions, different amounts of paraffin/wax may be deposited onto the surface of the cell. In some aspects, relatively light deposition may be sufficient for a protective effect. In other aspects, a relatively moderate deposition may be sufficient for a protective effect. In still other aspects, relatively heavy deposition may be sufficient for a protective effect. In yet other aspects, wherein a plurality of probes are used, each type of probe may generate a light deposition of paraffin/wax that in combination with depositions from other probes are sufficient for a protective effect.

[00033] In some embodiments, the probe comprises an antibody to a specific antigen, with the antibody complexed or conjugated to a moiety capable of directing a conjugate of paraffin/wax to form a protective barrier. In another embodiment, the probe comprises an antibody to a specific antigen, with the antibody directly conjugated to or coated with paraffin/wax. In still another embodiment, the probe comprises a primary antibody that binds to a tissue antigen and a second antibody that binds to the primary antibody, where the secondary antibody is covalently conjugated or non-covalently complexed to a moiety capable of directing deposition of paraffm/wax to form a protective barrier.

[00034] As an alternative, enzyme inhibitors (e.g., inhibitors to the enzyme used for degradation, such as proteinase K, trypsin, etc.) or other inhibitors of chemical processes may be complexed or conjugated to the probe, e.g., via the secondary antibody. In this embodiment, the inhibitor may locally deactivate or inhibit the degradation or processing enzyme or other reagent so that target cells covered by the probe are protected.

[00035] Essentially any antibody may be selected as a primary antibody, provided that it specifically binds to an antigen associated with a target cell type or subcellular structure. Methods of determining antibody specificity are well known in the art. An antibody typically is considered specific if it binds to a target antigen without binding to unrelated antigens. In some embodiments, the primary antibody may bind to an antigen associated with a type of normal cell. In other embodiments, the primary antibody may bind to an antigen associated with a type of cancerous or tumorigenic cell. In still other embodiments, the primary antibody may bind to an antigen associated with a type of immune cell.

[00036] In some aspects, the antibodies may bind to antigens present on the surface of the cell, to extracellular antigens, or antigens present within the interior of the cell (provided that the cell was cross-sectioned during slide preparation).

[00037] Examples of suitable antibodies include, but are not limited to: cytokeratin AE1/AE3 antibodies, anti-Ki-67 antibodies (Ki-67 is a nuclear antigen), anti-histone antibodies (histone is a nuclear antigen), anti-Epithelial Membrane Antigen (EMA) antibodies (EMA is a cell membrane antigen), anti-CD8 antibodies (CD8 is a lymphocyte antigen), anti CD20 antibodies (CD20 is a lymphocyte antigen), anti-vimentin antibodies (vimentin is a stroma antigen), etc. In some aspects, cytokeratin AE1/AE3 antibodies may be a mixture of two different clones of anti-cytokeratin monoclonal antibodies, e.g., AE1 that detects high molecular weight keratins and AE3 that detects low molecular weight keratins. In other aspects, antibodies to mitochondrial antigens, antibodies to Golgi antigens, antibodies to nuclear antigens may be generated and used with the methods and systems provided herein. [00038] Either a single probe or a cocktail of multiple probes can be utilized to protect cells, allowing flexibility and tunability with regard to experimental strategy. For example, in a histological section containing cancer cells, a probe can target the tumor cells during the micropurification process, thus separating the slide into two components; neoplastic cells versus all of the normal cell types (e.g., fibroblast, nerve, lymphocyte, etc.) and structural components (e.g., basal lamina, matrix, neo-vessels, etc.) present in the tumor microenvironment. If the probe(s) targets tumor cells, then the normal cells and structural components are solubilized or otherwise processed, leaving behind the tumor cells. If a cocktail of probes target normal cells and structural components, then the tumor cells are solubilized or processed.

[00039] Suitable titers for antibodies include 1 : 10, 1 :20, 1 :50, 1 :100, 1:200, 1 :500, 1 : 1000, 1 :2500, or any other amount.

[00040] In general, for antibody binding, any suitable incubation time may be used, provided that antigens exposed by antigen retrieval remain exposed and in a form in which antibody recognition and binding of the probe may occur. Examples of suitable incubation times for primary and/or secondary antibodies include, but are not limited to, about 15 minutes, about 30 minutes, about 45 minutes, about 60 minutes, or any range in between. In other aspects, incubation times with the secondary antibody may be about 15 minutes, about 30 minutes, about 45 minutes, about 60 minutes, or any range in between.

Probes - Affimers

[00041] As another example, an affimer may be used as a probe. Affimers are small domain proteins that specifically bind to an antigen and typically exhibit high thermal and biophysical stability, and typically lack di-sulfide bonds or other post-translational modifications.

[00042] Affimers may be utilized with the HMP methods described herein. In some aspects, affimers may be biotinylated and direct deposition of a paraffin/wax barrier using streptavi din-conjugated paraffin/wax. In general, any suitable reporter molecule may be used.

Probes - DNA [00043] A further type of probe that may be used in the methods described herein is an in situ hybridization (ISH) probe. ISH uses a complementary DNA or RNA strand to localize to a specific DNA or RNA sequence in a tissue specimen and may be used to determine whether genetic mutations (e.g., gene deletion, chromosome translocation, gene amplification, etc.) are present. A combination of one or more antibody probes and one or more ISH probes allows identification and separation of cells and tissues using the methods and systems described herein that previously were difficult to visualize.

[00044] For example, a DNA probe may be generated that is complementary to a target DNA sequence of interest. The DNA probe may be labeled, for example with a digoxigenin label. A primary antibody can be used to bind to the probe (e.g., the primary antibody may be an anti-digoxigenin fluorescein isothiocyanate antibody). A secondary antibody may bind to the primary antibody, wherein the secondary antibody contains a reactive moiety capable of directing deposition of a protective paraffm/wax barrier at the target site.

[00045] In general, the probe may comprise any molecule that binds to a target molecule, wherein the probe can direct deposition of a protective paraffm/wax barrier onto labeled cells.

Protective Barrier Material

[00046] In embodiments of the methods described herein, a hydrophobic protective barrier is deposited or placed on the surface of the target cells labeled with the probe. In some embodiments, the material that forms the hydrophobic protective barrier may be attached to the probe or may be attached to subsequent binding partners such as for example a secondary antibody. The protective material may be soluble or insoluble.

[00047] In this case a secondary antibody may be conjugated to a moiety such as biotin, which when bound to the primary antibody provides a mechanism to deposit streptavidin -conjugate paraffm/wax to the targets bound by the primary antibody.

[0048] The methods and systems provided herein are intended to provide capability of locally depositing a hydrophobic protective barrier consisting of paraffm/wax in a cell-specific manner that may be used with the micropurification techniques provided herein.

[00049] In other aspects, the present techniques may be used for ligand receptor binding studies. For example, the precision of the HMP process may be used to localize the protective effect to selected ligand and/or bound receptors in a specific subcellular region, recovering only the biomolecules (e.g. signaling molecules) in the immediate vicinity that are associated with the receptor.

Tunability

[00050] The methods and systems provided herein are highly tunable, and may be configured to isolate a wide variety of targets, including types of normal cells, types of diseased cells (e.g., such as a cancerous or tumorigenic cell), as well as immune cells. Once isolated, the cells may be subjected to further analysis, including: NGS, expression profiling, proteomic analysis, lipid analysis, etc. In general, any target for which a suitable probe is provided, e.g., a probe which can specifically bind to the target, and which can react with a reagent to direct deposition of a hydrophobic protective barrier, e.g., paraffm/wax to cover the cell, may be isolated by the methods and systems provided herein.

[00051] In particular, the components of this process (e.g., probe, conjugated paraffm/wax, micropurification solution, buffers, etc.) may be individually tuned to a particular assay, and once the assay has been validated, the identified conditions (e.g., temperature, time, antigen retrieval processing, etc.) may be further optimized.

Micropurification solution

[00052] The micropurification solution may be used to degrade, digest or otherwise process cells that have not been protected by the hydrophobic protective barrier. The solution may contain at least one protease and, optionally, a detergent, and other enzymes and chemical reagents. For example, the micropurification solution, also referred to as a digestion buffer, may contain 1 mg/ml proteinase K and 2% SDS. When present, the SDS concentration may be from 0.01% SDS up to 5% SDS, or any amount in between.

[00053] The concentration of the protease used in the micropurification can be any concentration sufficient to degrade or process the cells not protected by the protective barrier. For proteinase K, the concentration may range from about 0.1 mg/ml to about 10 mg/mg, from about 0.5 mg/ml to about 5 mg/ml, or any amount in between. In some aspects the concentration of proteinase K is about 1 mg/ml , about 2 mg/ml, or about 3 mg/ml.

[00054] The slide is incubated in the digestion buffer for a time sufficient to digest the cells not protected with the hydrophobic protective barrier to the extent necessary to allow removal or processing of those cells. For example, the incubation time may be about 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes, 75 minutes, 90 minutes, 120 minutes or 150 minutes or any amount in between. For longer incubation times, additional buffer or diH2O may be added so that the slides do not dry out. In some aspects, digestion/ processing during micropurification may be complete in 10-20 minutes.

[00055] In general, any serine protease may be used, including but not limited to, proteinase K, trypsin, chymotrypsin, etc. In some embodiments, the micropurification solution comprises one serine protease, or combinations of two serine proteases, or three or more serine proteases. In still other embodiments, degradation enzymes may be selected based upon the type of tissue to be analyzed, e.g., enzymes known to degrade muscle tissue and cells may be selected for slides containing muscle tissue, enzymes known to degrade adipose tissue and cells may be selected for slides containing adipose tissue, etc.

[00056] In general, any suitable detergent may be used, including but not limited to SDS, ADS, Triton X-100, Sarkosyl, Tween, etc. Further, in some aspects, two or more detergents may be used as part of the micropurification solution.

[00057] In general, any suitable buffer may be used, including but not limited to Citra Plus, Tris-HCl, citrate buffer, etc. Further, in some aspects, two or more buffers may be used as part of the micropurification solution.

[00058] The pH of the micropurification solution may range anywhere from a pH of 1 to a pH of 10. In general, the pH should match the optimal conditions of the micropurification enzyme.

[00059] In some embodiments, the hydrophobic protective barrier may prevent, inhibit, or reduce degrading components present in the micropurification buffer (e.g., proteases, detergents, etc.) from physically contacting the targeted cells. In other embodiments, the hydrophobic protective barrier may act to inhibit or reduce activity of a degrading enzyme, e.g., such as a protease. In still other embodiments, the hydrophobic protective barrier may restrict or confine motion of the protected biological material to prevent or reduce exposure to the degrading enzymes. [00060] In some aspects, the techniques and systems provided herein may be used to identify novel, general, or patient specific biomarkers. For example, analysis of the digested, processed, or solubilized portion of the slide may lead to the identification of novel serum biomarkers or validation of predicted biomarkers. Solubilized protein from microprotected cells can be analyzed via SDS-PAGE, western blot, reverse phase protein arrays, dot blots, mass spectrometry, etc.

Kits

[00061] Kits may be provided to carry out the methods presented herein. The HMP kit may comprise any or all of the reagents necessary to carry out the methods described herein. The kit may comprise a micropurification solution, and optionally, a neutralization solution that deactivates enzymes present in the micropurification solution to halt micropurification. In other embodiments, the kit may comprise individual reagents which are combined to form the micropurification solution.

[00062] In some aspects, the kit may comprise a probe, or a cocktail of more than one probe, and conjugated paraffin/wax which reacts with the probe, or multiple probes, to generate a hydrophobic protective barrier, along with the micropurification solution. The probe may be a monoclonal antibody, or a cocktail of multiple probes, or other antigen binding molecule or molecules, which specifically binds to an antigen present in the biological sample. The probe may be conjugated to a moiety capable of reacting with the conjugated paraffin/wax to produce the hydrophobic protective barrier.

[00063] Kits also are provided comprising the micropurification solution, a second antibody or second antigen binding fragment thereof that specifically binds to a first monoclonal antibody or first antigen binding fragment thereof. In this example, the first monoclonal antibody or first antigen binding fragment may be custom generated (not part of the kit) and designed to bind to the second antibody or second antigen binding fragment.

Custom mAbs may be produced, e.g., to bind to a specific marker (or markers) associated with a cell, depending on the nature of the assay. The second antibody or second antigen binding fragment is conjugated to or complexed with a moiety, which reacts with the paraffin/wax conjugate to produce a hydrophobic protective barrier. For example, the second monoclonal antibody may be biotinylated to facilitate binding of streptavidin conjugate paraffin/wax, where the paraffin/wax forms the hydrophobic protective barrier. In other aspects, the probe may additionally comprise a first monoclonal antibody or other antigen binding fragment, specific to a molecule present in the biological sample.

[00064] In some aspects, it may be desirable to inactivate proteinase K. The kit may include a neutralization solution comprising a protease inhibitor such as PMSF or AEBSF (Pefabloc®) to permanently inactivate proteinase K. In some embodiments, the kit may include a processing step of heat (e.g., 95°C for about 10 minutes) to inactivate proteinase K. [00065] In other embodiments, a kit may contain one or more of the following reagents for immunostaining and microprotection, including:

[00066] Reagent 1: Non-Aqueous Mounting Medium (e.g., Xylene)

[00067] Reagent 2: Serum Blocking Reagent

[00068] Reagent 3: Primary Antibody Diluent Reagent (may comprise BSA)

[00069] Reagent 4: Biotinylated Secondary Antibody

[00070] Reagent 5: Streptavi din-Conjugated paraffin/wax

[00071] Reagent 6: primary antibody-conjugated paraffin/wax

[00072] Reagent 7: paraffin/wax-conjugated Secondary Antibody

[00073] Reagent 8: antibody incubation solutions

[00074] Reagent 9: Counter stain (e.g., hematoxylin)

[00075] Reagent 10: Micropurification Solution

[00076] Reagent 11 : Micropurification Neutralization Solution

[00077] Reagents may be provided in concentrated form. In some embodiments, a primary mAb may be provided with one or more of Reagents 1-11. In other embodiments, a primary mAb may be provided with one or more of Reagents 2-8.

[00078] The HMP kit for biomolecule analysis (e.g., including analysis for DNA, RNA, lipid, protein, metabolite, etc.) from cells obtained from clinical or animal model tissue specimens will at least comprise a micropurification solution. Depending upon the usage for the particular type of kit, additional reagents may be provided to facilitate analysis of a particular biological sample, e g., DNA, mRNA, miRNA, and proteomic kits, which may or may not be optimized according to the techniques provided herein. Solutions and protocols optimized for each biomolecule and application are provided herein.

[00079] In other aspects, the HMP kit can be used with commercial IHC kits. For instance, IHC may be carried out using a commercial kit (e.g., http://zyagen.com/userfiles/Zyagen%20Peroxidase%201HC%20Kits.pdf), and once staining is complete, the micropurification solution may be added to capture target and non-target cells.

TaqMan™ assays

[00080] Once isolated, the target cells may be subject to further analysis, e.g., DNA analysis or gene expression assays, such as TaqMan® DAN and RNA assays. A variety of TaqMan® gene sequence and gene expression assays are available (e.g., ThermoFisher's TaqMan® Gene Expression Assay, https://www.thermofisher.com/us/en/home/life- science/pcr/real-time-pcr/real-time-pcr-assays.html?ef_id=fabld649ffacl7467ac7b054a00c 5bdO:G:s&s_kwcid=AL!3652! 10!76965986207600!76966046110358&cid=gsd_pcr_sbu_r02_co _cpl491_pjt9621_gsd00000_0se_bng_rs_pur_). Nucleotide hybridization probes may be obtained that specifically hybridize to DNA comprising the mutation of interest. In other aspects, custom hybridization probes may be designed to screen for particular mutations.

Advantages

[00081] In addition to being widely accessible due to its simplicity and low cost, the methods described herein provide a significant improvement in precision compared to current methods. While laser dissection instruments have been used to perform ultra-precise procurement in the past, e.g., recovering individual nuclei, this is still an extremely challenging undertaking that typically retrieves only a few targets due to the tediousness of the human-based targeting process. For most downstream molecular assays, procuring a large number of cells or organelles is advantageous [23, 35, 36, 47-49], since the reproducibility and robustness of data sets generally improve as the amount of starting material increases.

[00082] Present techniques allow for moderate- and low-abundant molecules (miRNAs, mRNAs) and DNA allele variants to be isolated and even measured. This is important for assays such as proteomic assays wherein the dynamic range of expression is many orders of magnitude. Because micropurification is based on a molecular probe for targeting, all stained cells, organelles, or nuclei are rapidly retrieved when the histological slide is processed, whether that is one cell/organelle/nuclei or one million cells/organelles/nuclei. Thus, relatively large amounts of DNA, RNA or protein from individual cells or from subcellular structures can be recovered for molecular analysis using the micropurification process.

[00083] Micropurification may be used to select and purify target and non-target cells, organelles and nuclei, as well as other structures, facilitating further analysis of a variety of different cell types and biological molecules including DNA, RNA, proteins, lipids, metabolites, etc. The protocols and components/reagents may be tuned and optimized to isolate a wide variety of different types of cells (e.g., normal and diseased), and biological molecules. Such techniques also may be used to quantitatively validate the robustness and reproducibility of mutation testing, and to extend measurement capability to labile components such as RNA, mRNA, and miRNA.

[00084] The methods and kits herein provide for a rapid, easy-to-use, precise (at a cellular level) and inexpensive cell procurement system that facilitates tumor mutation analysis for patient care and personalized medicine. These methods also are suitable for laboratory-based research, and do not rely upon sophisticated instrumentation, such as that found with laser dissection instruments. Accordingly, the methods may readily be performed in a variety of settings, including research labs, field applications, remote medical locations, etc.

[00085] In other aspects, the methods provided herein may be used to test for the presence of a variety of different types of tumor mutations. In some aspects, a series of histology samples may each be screened for the presence of a particular tumor marker. In other embodiments, cancer cells may be isolated, and the corresponding genetic material subjected to NGS to determine a mutation profile for a particular patient having a particular type of cancer.

[00086] In some aspects, processing may occur in parallel to facilitate high throughput analysis. Thus, in some aspects, a plurality of slides may be individually screened for the presence of a mutation or a protein in a manner of minutes (e g., in about 10-20 minutes or more). The isolated cells having the protein or mutation may undergo further processing. [00087] In some aspects, the methods provided herein may be performed manually. In other aspects, the methods may be incorporated into an automated system for processing histology slides, as human intervention or complex instrumentation is not needed to isolate target cells.

[00088] The HMP methods provided herein may be combined with microdissection, genomic analysis, and proteomic analysis. Such techniques can be used to facilitate personalized medicine, e.g., to quickly isolate a type of patient cell having a particular mutation or expressing a particular protein, wherein the isolated cells can undergo additional types of analysis, e.g., NGS, expression analysis, expression profiling, proteomic analysis, etc., to generate a personalized profile for a particular patient.

[00089] Other embodiments tailored to RNA studies may be developed using the techniques provided herein. While antigen retrieval may make non-target cells more susceptible to complete digestion and removal from the slide, this process may also hydrolyze RNA of both the target and non-target cells. As an alternative method, the micropurification solution may comprise RNase as the degrading enzyme to digest RNA in the non-target cells. For example, the hydrophobic protective barrier will prevent RNase from degrading RNA of protected cells. The protected cells may then be collected, and their respective RNA obtained for further analysis. In this example, DNA, proteins, or other materials present in the non-target cells do not need to be removed (i.e., complete digestion is not needed) since the downstream molecular assay for RNA analysis is specific to RNA.

[00090] Specific examples of HMP protocols are provided below. These examples are not inclusive of all HMP embodiments and are intended only to show the practicality of the invention. While the present invention has been described in terms of particular embodiments and applications, it is not intended that these descriptions in any way limit its scope to any such embodiments and applications, and it will be understood that many substitutions, changes and variations in the described embodiments, applications, and details of the method and system illustrated herein and of their operation can be made by those skilled in the art without departing from the spirit of this invention.

[00091] In some embodiments, the digestion/degradation step may be omitted, i.e. placing the slide in the micropurification buffer, as it may not be desirable to digest/degrade the non-target cells. In such applications, the hydrophobic protective barrier may act as a barrier or inhibitor to protect the labeled cells from any of: a toxin to kill live cells, an endonuclease or DNase to degrade DNA, a RNase to degrade mi/mRNA, tyramide labeling, other proteinases, chemical cleavage of proteins, a lipase to degrade fat, etc. Thus, the present techniques may be used to provide a hydrophobic protective barrier for any of a variety of conditions and reasons.

[00092] In general, it is to be appreciated that the methods and techniques presented herein are not limited to one specific set of experimental conditions, e.g., with regard to heating times, cooling times, buffers, reagent concentrations, other processing conditions, and may include a range of conditions and reagents suitable for achieving the desired effect of microprotection of target cells and purification/analysis. All such conditions and reagents are considered to be within the scope of the present application.

EXAMPLES

Example 1; Methods of Micropurification of Non-Target Cells

[00093] Example protocols for analyzing cells are provided herein. This example relates to FIGS. 1-4 for protective barrier deposition and FIG. 6 for cell procurement. An immuno-based histology procedure that places a hydrophobic protective barrier on target cells is provided herein, whereby the hydrophobic protective barrier deposition is directed by a probe, such as an antibody or aptamer, and protects a target cell population from a micropurification solution but does not protect non-target cells. Enzymatic treatment of the tissue section with the protective barrier present on target cells digests the non-target cells into the micropurification solution (digestion buffer). The slide may be washed, and the non-target cells present in the micropurification solution can be either retrieved from the slide for molecular analysis or discarded and not analyzed.

Preparation

[00094] In some cases, H&E stains are initially performed on tissue sections to assess morphology and cellular content. The microtissue slide is placed in xylenes for about 10 minutes, and was rehydrated using graded alcohols and water. Slides are placed in a staining rack within a Tissue Tek staining dish, filled with 250ml of lx Citra Plus buffer (BioGenex), and covered loosely.

Antigen Retrieval

[00095] After preparation, antigen retrieval is performed. Antigen retrieval involves exposing the surface of the cells, e.g., by melting and removing polymer that may cover the cells during paraffin embedding and fixation of the biological sample onto the histology slide. Once exposed, antibodies or other molecular probes may contact the biological sample, e.g., to bind to markers, proteins, receptors, nucleotide sequences, etc. For slides embedded in paraffin, antigen retrieval may help induce or enhance susceptibility of the sample to micropurification. [00096] The slides, covered by Citra Plus buffer, are microwaved for 5 minutes at high power (Sanyo, 1200 Watts) until boiling. The buffer level is checked and diH2O is added, as needed, to keep the tissue sections moist throughout the procedure. The slides are then microwaved for 15 minutes at power level 3, the buffer level is inspected halfway through the 15 minutes, and diH2O can be added as needed. The slides are then allowed to cool in the microwave for an additional 20 minutes. Finally, the slides are rinsed with diH2O. Hydrophobic Protective Barrier Deposition

[00097] Following antigen retrieval, the slides are incubated at room temperature for about two hours with a primary antibody, for about one hour with a secondary antibody conjugated to a biotin molecule(s), and then deposition of the hydrophobic protective barrier can be completed by incubating the slide at room temperature for 1 hour with streptavidin- conjugated paraffin/wax (FIG. 1). The primary antibodies used as the probe can be cytokeratin AE1/AE3 mAbs (1 : 100, BioGenex) and anti-Ki-67 mAbs (1: 100, BioGenex) for protection of tumor cells in a section of colon cancer tissue. The slide is then rinsed in water to remove the unbound streptavi din-conjugated paraffin/wax. After paraffin/wax deposition mediated by the interaction of the biotin molecule on the biotinylated secondary antibody with the streptavidin molecule conjugated to paraffin/wax, slides are heated to a temperature above the melting point of the paraffin/wax (for example 65°C) for 1 minute to infuse the melted paraffin/wax into, around, and on top of the target cells followed by removing the slide from heat and cooling to room temperature. The hydrophobic protective barrier is now deposited on desired target cells as mediated by the probe (primary antibody), whereby the paraffin/wax becomes a solid hydrophobic protective barrier upon cooling below the melting point of the paraffin/wax. Micropurification

[00098] Slides are incubated in 200-300 microliters of micropurification solution

(also referred to as digestion buffer) containing for example 1 mg/ml proteinase K and 0.1% SDS for 10 mins at 56°C. The digestion step dissolved away the non-protected cells and extracellular structures. The micropurification solution that now contains digested non-target cells along with all the molecules that make up cells including DNA is removed into a separate tube. In this way the non-target cells are recovered from the slide and can be molecularly analyzed, as for example using polymerase chain reaction and next generation sequencing to analyze DNA and RNA present in the non-target cells (FIG. 5).

[00099] It is noted that the target cells remain on the slide because they are protected from digestion by the micropurification solution. These target cells can then be collected following the procedure outline below in Example 2.

Molecular Analysis

[000100] DNA can be extracted from the recovered non-target cells via overnight incubation with proteinase K at 65°C. Samples are heated at 95°C for 15 minutes to inactivate proteinase K. Prepared DNA can be purified by simple spin column and eluted into a new tube in a concentrated and purified form. This DNA is then ready for PCR and/or NGS analysis.

[000101] Similar techniques may be employed to recover and analyze additional biomolecules in the target cell(s)/structure, including RNA, proteins, lipids, metabolites, and others. For example, the isolated cells may be subjected to RNA analysis, e g., using one of many different RNA preparation kits. In other aspects, the isolated cells may be subjected to protein analysis, e.g., using mass spectrometry proteomics. In still other aspects, Abcam's lipid extraction kit may be used to isolate lipids for analysis by mass spectrometry lipidomics methods. Accordingly, once isolated, the cells may be subjected to a variety of different types of analysis to recover and analyze biomolecules in the target cell(s)/structure, including RNA, proteins, lipids, metabolites, and so forth.

Example 2; Methods of Purification of Target cells

[000102] An example protocol for collecting and analyzing protected target cells is provided herein (FIG.6). The protocol begins with the same slide whereby the non-protected cells have been removed and collected for molecular analysis using the micropurification solution (Example 1 and FIG. 5).

Barrier Removal

[000103] Once the non-protected cells have been removed through the process of micropurification of the non-target cells, the target cells can be collected. This can be performed by first removing the hydrophobic protective barrier by standard methods of dewaxing a histological slide. This is performed simply by incubating the slide in xylenes for about 10 minutes followed by rehydrating the slide using graded alcohols and water. In this way the paraffin/wax that formed the hydrophobic protective barrier is dissolved away by the xylenes exposing the target cells that can now be removed by micropurification and analyzed using the same methods as used for analyzing the non-target cells.

Micropurification

[000104] Slides are incubated in 200-300 microliters of micropurification solution

(also referred to as digestion buffer) containing for example 1 mg/ml proteinase K and 0.1% SDS for 10 mins at 56°C. The digestion step dissolves into the micropurification solution the now- exposed, now non-protected target cells. The micropurification solution that now contains digested target cells along with all the molecules that make up the target cells including DNA is removed into a separate tube. In this way the target cells are recovered from the slide and can be molecularly analyzed, as for example using polymerase chain reaction and next generation sequencing to analyze DNA and RNA present in the non-target cells (FIG. 6).

Molecular Analysis

[000105] DNA is extracted from the recovered target cells via overnight incubation with proteinase K at 65°C. Samples are heated at 95°C for 15 minutes to inactivate proteinase K. Prepared DNA can be purified by simple spin column and eluted into a new tube in a concentrated and purified form. This DNA is then ready for PCR and/or NGS analysis.

[000106] Similar techniques may be employed to recover and analyze additional biomolecules in the target cell(s)/structures, including RNA, proteins, lipids, metabolites, and others. For example, the isolated and collected cells, and or subcellular structures, may be subjected to RNA analysis, e.g., using one of many different RNA preparation and analysis kits and procedures. In other aspects, the isolated and collected target cells may be subjected to protein analysis, e.g., using mass spectrometry proteomics. In still other aspects, one of many lipid extraction kits may be used to isolate lipids for analysis by mass spectrometry lipidomics methods. Accordingly, once isolated, the cells or subcellular structures may be subjected to a variety of different types of analyses to recover and analyze biomolecules in the target cell(s)/structure(s), including RNA, proteins, lipids, metabolites, and so forth.

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Claims

1. A method of collecting cells from a biological sample comprising the following steps: bind a specific probe to specific target cells, cellular structures, subcellular structures, and/or tissue structures to localize and direct the deposition of a protective hydrophobic barrier onto said specific target cells, cellular structures, subcellular structures, and/or tissue structures present in a histological sample affixed to a support surface comprising cells, cellular structures, subcellular structures, and/or tissue structures infused with, surrounded by, and covered with said protective barrier, and apply a liquid micropurification solution to the biological sample present in a histological sample affixed to a support surface, wherein said liquid micropurification solution degrades, digests, or otherwise differentially processes cells, cellular structures, subcellular structures, and/or tissue structures not protected by the said solid, bound, protective hydrophobic complex comprising cells, cellular structures, subcellular structures, and/or tissue structures infused with, surrounded by, and covered with said protective barrier, and collect the protected target cells, cellular structures, subcellular structures, and/or tissue structures for molecular analysis and/or collect the unprotected cells, cellular structures, subcellular structures, and/or tissue structures for molecular analysis.

2. The method of claim 1, wherein a plurality of specific probes is used, and wherein each probe binds to a different target.

3. The method of claim 1 or 2, wherein said probe comprises a primary antibody and a secondary antibody, wherein said primary antibody specifically binds specific cells, cellular structures, subcellular structures, and/or tissue structures in said tissue, and wherein said secondary antibody specifically binds said primary antibody and comprises a reactive moiety capable of reacting with said hydrophobic protective barrier material to form said barrier.

4. The method of claim 3, wherein said antibody binds to a tumor marker, a cancer marker, a nuclear marker, an extracellular marker, an intracellular marker, or an immunological marker.

5. The method of claims 1-3, wherein said hydrophobic protective barrier material comprises wax, paraffin, paraffin wax, petroleum wax, or paraffin/wax -based compositions.

6. The method of claim 1, wherein said protective barrier material is localized to and delivered to the target cells, cellular structures, subcellular structures, and/or tissue structures as directed by the presence of a probe bound to targeted cells, cellular structures, subcellular structures, and/or tissue structures at a temperature below the melting point of the hydrophobic protective barrier material.

7. The method of claim 1, wherein said hydrophobic protective barrier material localized to the target cells, cellular structures, subcellular structures, and/or tissue structures by the presence of a probe bound to targeted cells, cellular structures, subcellular structures, and/or tissue structures is heated to a temperature above the melting point of the protective barrier material for an approximate time of 10 seconds to 10 minutes, thus liquifying the hydrophobic protective material.

8. The method of claim 1, wherein said heated and liquified protective barrier material deposited as directed by the presence of a probe bound to target cells, cellular structures, subcellular structures, and and/or tissue structures is incubated at a temperature below the melting point of the protective barrier material to form a solid, bound, protective hydrophobic complex within, around, and above said target cells, cellular structures, subcellular structures, and/or tissue structures within a histological sample affixed to a solid surface.

9. The method of claim 1, wherein the location or locations of contacting a histological sample affixed to a surface with a hydrophobic protective barrier material is directed by the presence of a probe and processes utilizing said probe, including but not limited to, immunohistochemistry (IHC), in-situ hybridization (ISH), histological staining reagents, a primary antibody, a secondary antibody, and/or an aptamer.

10. The method of claim 1, further comprising collecting cells, cellular structures, subcellular structures, and/or tissue structures protected from the micropurification solution by the solid, bound, hydrophobic protective complex, wherein the cells collected are selected from the group consisting of neoplastic cells, cancer cells, tumor cells, immune cells, normal cells, subcellular compartments, subcellular structures, and structural components of a tissue.

11. The method of claim 1, further comprising: isolating DNA from the collected cells and determining a DNA allelotype; or analyzing the collected cells using a method comprising one or more of next generation sequencing (NGS), expression profiling, proteomic analysis, lipid analysis, metabolism analysis, methylation analysis, immunohistochemical staining, histological staining, and/or immunological activity analysis.

12. The method of claim 1, wherein the histological sample affixed to a solid surface is embedded in paraffin.

13. The method of claim 1, wherein said histological sample affixed to a surface is formalin fixed.