Chromogenic multiplexing methods and systems

Abstract

A method and apparatus for labeling a tissue section is provided. In certain aspects, the methods comprise labeling a tissue sample via a plurality of immunohistochemistry (IHC) assays for detection of markers for characterization of a non-Hodgkin's lymphoma. The disclosed IHC assays employ chromogen-based detection methods for improved sample efficiency and visualization of biomarkers. Further disclosed is an apparatus for carrying out the disclosed methods.

Description

LBR 20276-P-US CHROMOGENIC MULTIPLEXING METHODS AND SYSTEMS BACKGROUND

[0001] Non-Hodgkin lymphoma (NHL) is a heterogeneous group of chronic lymphoproliferativedisorders originating in the B, T, or natural killer (NK) lymphocytes, characterized by abnormal proliferation of monoclonal lymphocytes in the peripheral blood, the bone marrow, the lymph node, or any other lymphoid tissue. NHL is a major health concern globally. Via detection of biological marker in the tissue, immunohistochemistry (IHC) is one technique used to characterize NHL. IHC utilizes antibody-based detection of biological markers in tissue which can be used for diagnosis, disease characterization, and / or for developing personalized treatment. While IHC to detect biomarkers is increasingly useful to diagnosing and treating patients with NHL, the increase in biomarkers being required for diagnosis and targeted care has led to an increasing amount of tissue being required for basic tumor characterization. Simultaneously, with advances in radiology and supporting technologies, biopsy samples are becoming smaller, making the availability of tissue samples limited. As such, there is an increased need for tissue samples and a simultaneous decrease in the amount of tissue sample available for such testing.

[0002] Multiplex methods, in which more than one detectable agent (e.g., antibody specific for abiological marker) is applied to a single sample, allows for detection of more than one biomarker in a single tissue sample and therefore has the benefit of reducing the amount of tissue sample needed. However, while such methods have the potential to reduce the amount of tissue needed to detect multiple targets, the effectiveness of tri-plexed (three-color applications) or greater has not been widely employed because certain chromogens have overlapping spectral properties, making it challenging to differentiate between signals and accurately quantify multiple analytes simultaneously.

[0003] As such, there is a need in the art for improved multiplex methods. The instant disclosureseeks to address one or more of the aforementioned needs in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0004] While the specification concludes with claims which particularly point out and distinctlyclaim this technology, it is believed this technology will be better understood from the followingLBR 20276-P-US description of certain examples taken in conjunction with the accompanying drawings, in which like reference numerals identify the same elements and in which:

[0005] FIG. 1 depicts a perspective view of an example of a slide-staining instrument;

[0006] FIG. 2 depicts a perspective view of a tray for use with the slide-staining instrument ofFIG.1;

[0007] FIG. 3 depicts another perspective view of the tray of FIG. 2 with a slide and slide coverloaded onto the tray;

[0008] FIG. 4 depicts a perspective view of a slide staining assembly of the slide-staininginstrument of FIG.1;

[0009] FIG. 5 depicts a flow chart of an example of an Immunohistochemistry (IHC) AssayProtocol for use with the slide-staining instrument of FIG.1;

[0010] FIG. 6 depicts a schematic diagram of a control system configured to implement theImmunohistochemistry Assay Protocol (100) of FIG.5 using the slide-staining instrument of FIG. 1.

[0011] FIGS. 7A-7C depict an exemplary result following the protocol of Example 1, Layer 1,demonstrating a clinical use tissue expression. FIG.7A depicts multiplex IHC staining using the disclosed methods on a tissue sample from an individual having Follicular Lymphoma (Ki67: Brown, CD3: Red, CD20: Green; FIG. 7B depicts multiplex IHC staining using the disclosed methods on a tissue sample from an individual having Mantle Cell Lymphoma (Ki67: Brown, CD3: Red, CD20: Green; FIG.7C depicts multiplex IHC staining using the disclosed methods on a tissue sample from an individual having Diffuse Large B Cell Lymphoma (Ki67: Brown, CD3 Red, CD20 Green).

[0012] FIGS. 8A-8C depict an exemplary result following the protocol of Example 1, Layer 2,demonstrating a clinical use tissue expression and co-localization using the disclosed methods. FIG.8A depicts multiplex IHC staining using the disclosed methods on a tissue sample from an individual having Follicular Lymphoma (MUM1: Brown, CD10: Red, CD5: Green; FIG. 8B depicts multiplex IHC staining using the disclosed methods on a tissue sample from an individualLBR 20276-P-US having Mantle Cell Lymphoma (MUM1: Brown, CD10: Red, CD5: Green; FIG. 8C depicts multiplex IHC staining using the disclosed methods on a tissue sample from an individual having Diffuse Large B Cell Lymphoma (MUM1: Brown, CD10: Red, CD5: Green).

[0013] FIG.9 is a multiplex IHC result showing co-localization of CD10 (Red) and CD5 (Green)which form a fourth color in a small subset of B cells. Representative co-localization events are circled.

[0014] FIGS. 10A-10C depicts an exemplary result following the protocol of Example 1, Layer3, demonstrating a clinical use tissue expression. FIG.10A depicts multiplex IHC staining using the disclosed methods on a tissue sample from an individual having Follicular Lymphoma (Bcl-2: Brown, Cyclin D1: Red, Bcl-6: Green; FIG. 10B depicts multiplex IHC staining using the disclosed methods on a tissue sample from an individual having Mantle Cell Lymphoma (Bcl-2: Brown, Cyclin D1: Red, Bcl-6: Green; FIG. 10C depicts multiplex IHC staining using the disclosed methods on a tissue sample from an individual having Diffuse Large B Cell Lymphoma (Bcl-2: Brown, Cyclin D1: Red, Bcl-6: Green).

[0015] The drawings are not intended to be limiting in any way, and it is contemplated thatvarious embodiments of the technology may be carried out in a variety of other ways, including those not necessarily depicted in the drawings. The accompanying drawings incorporated in and forming a part of the specification illustrate several aspects of the present technology, and together with the description explain the principles of the technology; it being understood, however, that this technology is not limited to the precise arrangements shown. DETAILED DESCRIPTION

[0016] The following description of certain examples of the technology should not be used tolimit its scope. Other examples, features, aspects, embodiments, and advantages of the technology will become apparent to those skilled in the art from the following description, which is by way of illustration, one of the best modes contemplated for carrying out the technology. As will be realized, the technology described herein is capable of other different and obvious aspects, all without departing from the technology. Accordingly, the drawings and descriptions should be regarded as illustrative in nature and not restrictive.LBR 20276-P-US I. DEFINITIONS

[0017] Unless defined otherwise herein, all technical and scientific terms used herein have thesame meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described.

[0018] All patents and publications, including all sequences disclosed within such patents andpublications, referred to herein are expressly incorporated by reference.

[0019] The headings provided herein are not limitations of the various aspects or embodimentsof the invention. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.

[0020] Unless defined otherwise, all technical and scientific terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The practice of the present technology will employ, unless otherwise indicated, conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology, and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual (Table of Contents available at www.cshlpress.com / pdf / sample / 2013 / MC4 / MC4FM.pdf).

[0021] All numerical designations, e.g., pH, temperature, time, concentration, and molecular5%, or alternatively 2%. It is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art.

[0022] It is to be inferred without explicit recitation and unless otherwise intended, that when thepresent technology relates to a polypeptide, protein, polynucleotide or antibody, an equivalent or a biologically equivalent of such is intended within the scope of the present technology.LBR 20276-P-US

[0023] A “diagnostic marker” is a specific biochemical in the body which has a particularmolecular feature that makes it useful for detecting a disease, measuring the progress of disease or the effects of treatment, or for measuring a process of interest.

[0024] The term “epitope” as used herein is defined as small chemical groups on the antigenmolecule that is bound to by an antibody. An antigen can have one or more epitopes. One skilled in the art understands that generally the overall three-dimensional structure or the specific linear sequence of the molecule can be the main criterion of antigenic specificity.

[0025] A “subject” of diagnosis or treatment is a plant or animal, including a human. Non-humananimals subject to diagnosis or treatment include, for example, livestock and pets. The term “individual” or “patient” is used interchangeably.

[0026] As used herein, the term “tissue section” refers to a piece of tissue that has been obtainedfrom a subject and mounted on a planar surface, e.g., a microscope slide. The sample may be fixed and / or sectioned as desired. A “tumor tissue sample” or “tumor tissue biopsy sample” includes cells derived from a tumor in a subject, e.g., a human subject having a malignancy. Such tissue samples are sometimes referred to simply as a “biopsy”.

[0027] As used herein, the term “formalin-fixed paraffin embedded (FFPE) tissue section” refersto a piece of tissue, e.g., a biopsy that has been obtained from a subject, fixed in formaldehyde (e.g., 3%-5% formaldehyde in phosphate buffered saline) or Bouin solution, embedded in wax, cut into thin sections, and then mounted on a planar surface, e.g., a microscope slide.

[0028] “Molecule of interest,” “target molecule,” “target,” and “target antigen” each refers to amolecule for which the presence, location and / or concentration is to be determined. Examples of molecules of interest include proteins and nucleic acid sequences.

[0029] The term “staining” includes binding a target (e.g., an antigen) in a cellular sample with atarget-specific binding agent (e.g., an antibody) and then detecting the presence of the target- specific binding agent on the cells of the cellular sample using a detectable label or chromogen. The detectable label can be directly conjugated to the target-specific binding agent (e.g., a primary antibody) or may be conjugated to a secondary reagent that binds specifically to an unlabeledLBR 20276-P-US target-specific reagent (e.g., a secondary antibody). In some cases, the target-specific reagent is itself detectable, and thus no additional attached label is needed.

[0030] A “chromogen” or “chromogenic compound” and the like is a substance that can beconverted into a colored compound under specific conditions, e.g., when acted upon by an enzyme or under specific chemical / reaction conditions. Examples of enzyme-substrate combinations include: (i) Horseradish peroxidase (HRP) with hydrogen peroxidase as a substrate, where the hydrogen peroxidase oxidizes a dye precursor; and ii) alkaline phosphatase (AP). Numerous other enzyme-substrate combinations are available to those skilled in the art. For a general review of these, see U.S. Pat. Nos.4,275,149 and 4,318,980.

[0031] As used herein, the term “target-specific binding agent” or “binding moiety” means anyagent that specifically binds to a target or analyte of interest, e.g., a target of interest that is present in a tissue section as described herein (e.g., a polypeptide). Examples of target-specific binding agents include antibodies, receptors, and ligands, or target-binding fragments thereof, polynucleotide probes, and the like.

[0032] As used herein, the term “multiplexing” refers to using more than one label, stain, and / orchromogen for the simultaneous or sequential detection and measurement of a target in a sample, e.g., a tissue section. As used herein, the term “triplexing” refers to using three labels, stains, and / or chromogens for the simultaneous or sequential detection and measurement of a target in a sample, e.g., a tissue section.

[0033] As used herein, the terms “antibody” and “immunoglobulin” are used interchangeably andare well understood by those in the field. Those terms refer to a protein consisting of one or more polypeptides that specifically binds an antigen. One form of antibody constitutes the basic structural unit of an antibody. This form is a tetramer and consists of two identical pairs of antibody chains, each pair having one light and one heavy chain. In each pair, the light and heavy chain variable regions are together responsible for binding to an antigen, and the constant regions are responsible for the antibody effector functions. These terms also include fragments of antibodies which retain specific binding to antigen or target, including, but not limited to, Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single-chain antibodies, bi-specificLBR 20276-P-US hybrid antibodies, and fusion proteins comprising an antigen-binding portion of an antibody and a non-antibody protein.

[0034] As used herein, the terms “primary antibody” and “secondary antibody” refer to differentantibodies, where a primary antibody is a polyclonal or monoclonal antibody from one species (rabbit, mouse, goat, donkey, etc.) that specifically recognizes an antigen (e.g., a biomarker) in a sample (e.g., a human tissue sample) under study, and a secondary antibody is an antibody (usually polyclonal) from a different species that specifically recognizes the primary antibody, e.g., in its Fc region.

[0035] The term “specific binding” refers to the ability of a binding agent to preferentially bindto a particular analyte that is present in a homogeneous mixture of different analytes. In certain aspects, a specific binding interaction will discriminate between desirable and undesirable analytes in a sample, in some aspects more than about 10 to 100-fold or more (e.g., more than about 1000- or 10,000-fold). II. Multiplex Methods for Detecting NHL Biomarkers in a Sample Characterization of non-Hodgkin’s Lymphoma

[0036] Non-Hodgkin lymphoma (NHL) encompasses a diverse group of cancers that arise fromlymphocytes. The disclosed methods relate generally to methods for characterizing non-Hodgkin’s lymphoma in an individual in need thereof, and systems and apparatuses for carrying out the disclosed methods and may be used to achieve a variety of outcomes related to the characterization of NHL.

[0037] In one aspect, the disclosed methods may be used to inform a treating physician, based onthe detection of markers in a tissue sample obtained from an individual, of the presence or absence of NHL, the type of NHL, the severity of the NHL, and / or for development of a personalized dosage or treatment regimen for the individual. The disclosed methods may be used in conjunction with other patient-derived data, which may include, for example, additional biomarker tests, such as those described in U.S. Patents 9,234,892, 10,041,949 and 10,370,698, patient symptoms, patient history, assessment of cellular structure, and the like.LBR 20276-P-US

[0038] In one aspect, the disclosed methods may be used in the process of diagnosis, stagedetermination, or treatment identification or a subset determination of NHL. Subsets of NHL may include Diffuse large B-cell lymphoma, Anaplastic large-cell lymphoma, Burkitt lymphoma, Lymphoblastic lymphoma, Mantle cell lymphoma, Peripheral T-cell lymphoma, Follicular lymphoma, Cutaneous T-cell lymphoma, Lymphoplasmacytic lymphoma, Marginal zone B-cell lymphoma, MALT lymphoma, Small-cell lymphocytic lymphoma, and combinations thereof.

[0039] In one aspect, the disclosed methods may be used to obtain biomarker data from a tissuesection comprising a tissue sample obtained from an individual, which may be used in the process of selecting one or more drugs and / or dosage (amount, length of administration, etc.) for treatment of the individual. In other aspects, the methods may be used to modify or personalize a treatment plan based upon the patient's individual biomarker profile, based on a determination of the presence, absence, or level of one or more biomarkers as disclosed herein.

[0040] In one aspect, the disclosed methods may be used, alone or in combination with otherpatient-derived data, to determine an appropriate treatment plan for an individual, wherein the disclosed methods are used to determine the subset of NHL, and, based on the determination of the subset of NHL, a therapy is selected from chemotherapy, immunotherapy, a targeted therapy directed to a dysregulated signaling pathway, radiation therapy, stem cell transplant, or combinations thereof. Tissue Sample and Preparation of Tissue Section

[0041] Tissue samples and preparation of tissue sections may be carried out according to methodsknown in the art. For example, tissue may be obtained from a subject for routine screening or from a subject that is suspected of having a NHL, and may be isolated from an individual, e.g., from a soft tissue or from a bodily fluid, or from a cell culture that is grown in vitro, from brain, adrenal gland, skin, lung, spleen, kidney, liver, spleen, lymph node, bone marrow, bladder stomach, small intestine, large intestine, and / or muscle. Tissue samples may be obtained using any method known in the art, for example, via tissue biopsy, scrape, lavage, and combinations thereof.

[0042] Preparation of tissue sections are known. In one aspect, the tissue section may comprise atissue sample that has been formalin fixed, paraffin embedded (FFPE) as described, for example,LBR 20276-P-US in U.S. Patent Application Publication No. 2020 / 0049599. In brief, a tissue sample may be contacted with a formalin fixation buffer, dehydrated, and embedded in paraffin. Sections may then be cut, for example in three-micron thick sections and placed on a glass slide for analysis using the disclosed methods and systems. Target Antigens

[0043] NHL diagnosis, differential diagnosis and classification are commonly based, in part, onimmunohistochemistry analysis. Various immunophenotypic biomarkers are routinely used, including T-cell markers (CD2, CD3, CD4, CD5, CD7, CD8, and T-cell receptors), B-cell markers (CD19, CD20, CD22, CD79, and Pax-5), cytotoxic markers (such as TIA-1, granzyme B, and perforin), germinal center markers (CD10, Bcl-6, and LMO2), and plasma cell markers (such as CD38, CD138). The disclosed methods employ a three-panel assay, each panel employing a triplex assay (three IHC assays per panel), which allows for the detection of nine biomarkers using three tissue sections, thereby reducing the amount of tissue sample needed to perform the assay. With reference to FIG. 5, the disclosed methods provide for Immunohistochemistry Assay Protocol (100), which comprises a first IHC assay (Layer 1 Staining Protocol (110)) for detection of a first target antigen, a second IHC assay (Layer 2 Staining Protocol (210)) for detection of a second target antigen, and a third IHC assay (Layer 3 Staining Protocol (310)) for detection of a third target antigen, as described herein. In particular, the disclosed methods provide for the detection of the following biomarkers in a tissue sample: Ki67, CD3, CD20, MUM1, CD10, CD5, Bcl-2, Cyclin D1, and Bcl-6. Such biomarkers are known in the art and are described in brief herein.

[0044] Ki67

[0045] Ki67 is a protein marker used widely in cancer research and pathology to assess cellproliferation, particularly in tumors. The name “Ki67” refers to the location in the cell cycle where this protein is present, specifically during the active phases of cell division (mitosis and cytokinesis). The more actively dividing cells are, the higher the percentage of Ki67-positive cells. In cancer pathology, the Ki67 labeling index (the percentage of Ki67-positive cells in a tumor sample) is often used as a prognostic indicator. High levels of Ki67 expression are associated with more aggressive tumors and poorer prognosis in various types of cancer. Ki67 may be detected viaLBR 20276-P-US any antibody or binding moiety specific for Ki67, including the Ki67 (k2) antibody / clone available from Leica Biosciences, Product Code PA0230.

[0046] CD3

[0047] CD3 is a pan T-cell marker expressed on most of the mature T / NK-cell lymphomas,except for anaplastic large cell lymphoma. CD3 is homogenously expressed in T lineage lymphoma, and commonly used as a marker. CD3 may be detected via any antibody or binding moiety specific for CD3, including the CD3 (LN10) antibody / clone available from Leica Biosciences, Product Code PA0230.

[0048] CD20

[0049] CD20 is a pan B-cell marker that is expressed on most of the mature B-cell lymphomas.It is a cell surface protein marker that may be used for the diagnosis and treatment of lymphoma, particularly B-cell lymphomas. Expression of CD20 may be used to identify B-cell lymphomas and to differentiate between various subtypes of B-cell lymphoma, such as diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, mantle cell lymphoma, and others. CD20 is homogenously expressed in B lineage lymphoma, and commonly used as a marker. CD20 may be detected via any antibody or binding moiety specific for CD20, including the CD20 (L26) antibody / clone available from Leica Biosciences, Product Code PA0230.MUM1

[0050] MUM1 is a protein marker that is used in the diagnosis and classification of lymphomas,particularly in the context of diffuse large B-cell lymphoma (DLBCL). MUM1 may be used to characterize lymphoma cells and distinguish between different subtypes of DLBCL. MUM1 may be detected via any antibody or binding moiety specific for MUM1, including the MUM1 (EAU32) antibody / clone available from Leica Biosciences, Product Code PA0129.

[0051] CD10

[0052] Neprilysin (CD10) is a cell surface zinc-dependent metalloendopeptidase expressed innormal and neoplastic hematopoietic and non-hematopoietic tissue. CD10 constitutes a canonical element of current subclassification and prognostic algorithms in diffuse large B cell lymphoma (DLBCL), where its expression generally predicts more favorable outcomes. CD10 may beLBR 20276-P-US detected via any antibody or binding moiety specific for CD10, including the CD10 (56C6) antibody / clone available from Leica Biosciences, Product Code PA0131 / PA0270.

[0053] CD5

[0054] CD5 is a protein marker for T-cells that is also expressed in two B-cell neoplasms: mantlecell lymphoma and lymphocytic leukemia. The expression of CD5 on B-cell neoplasms is usually indicative of chronic lymphocytic leukemia (CLL) or mantle cell lymphoma (MCL). CD5 may be detected via any antibody or binding moiety specific for CD5, including the CD5 (4C7) antibody / clone available from Leica Biosciences, Product Code PA0168.

[0055] Bcl-2

[0056] Bcl-2 is an anti-apoptosis factor that is important in normal B-cell development anddifferentiation. Bcl-2 expression is a prognostic marker for activated B-cell-like diffuse large B- cell lymphoma. Bcl-2 may be detected via any antibody or binding moiety specific for Bcl-2, including the Bcl-2 (100 / D5) antibody / clone available from Leica Biosciences, Product Code PA0117.

[0057] Cyclin D1

[0058] Cyclin D1 is a useful marker for distinguishing mantle cell lymphoma from other types ofsmall B-cell lymphoma. Cyclin D1, for example, is overexpressed in lymphomas with (11;14)(q13;q32) translocation and is a diagnostic hallmark of mantle cell lymphoma. Cyclin D1 may be detected via any antibody or binding moiety specific for Cyclin D1, including the Cyclin D1 (EP12) antibody / clone available from Leica Biosciences, Product Code PA0046.

[0059] Bcl-6

[0060] Bcl-6 encodes a transcriptional factor that has a key role in germinal center B-celldifferentiation and in the pathogenesis of germinal center-derived lymphomas. Bcl-6 protein expression, a marker of germinal center origin, has been associated with a favorable prognosis in diffuse large B-cell lymphoma. Bcl-6 may be detected via any antibody or binding moiety specific for Bcl-6, including the Bcl-6 (LN22) antibody / clone available from Leica Biosciences, Product Code PA0204.LBR 20276-P-US Immunohistochemistry Methods

[0061] The disclosed methods generally use known principles of immunohistochemistry, inwhich a target antigen is bound to a binding moiety, in particular an antibody. “Histochemistry” and “cytochemistry” are techniques often used to identify biomarkers within the context of intact cells by labeling the samples with molecules that bind specifically to the biomarker in a manner that can be visualized on a microscope. Immunohistochemistry (IHC) and immunocytochemistry (ICC) are types of histochemistry and cytochemistry that use antibodies to label the biomarkers. By identifying the biomarker in the context of a tissue environment or cellular environment, spatial relationships between the biomarkers and other morphological or molecular features of the cell or tissue sample can be elucidated, which may reveal information that is not apparent from other molecular or cellular techniques. Immunohistochemistry methods are particularly useful to lymphoma diagnosis because the lymphocyte type (B or T cell) is indistinguishable on hematoxylin and eosin (H&E) stained slides. The technique is performed for diagnosis because cell lineage and maturation phase can be identified, specific genetic alterations can be detected, the degree of cell proliferation can be visualized, and therapeutic targets can be identified. (See, e.g., Cho, Junhun. “Basic immunohistochemistry for lymphoma diagnosis.” Blood research vol. 57,S1 (2022): 55-61).

[0062] Exemplary methods of immunohistochemistry are described in, for example, Magaki S,Hojat SA, Wei B, So A, Yong WH. An Introduction to the Performance of Immunohistochemistry. Methods Mol Biol.2019;1897:289-298. doi:10.1007 / 978-1-4939-8935-5_25. Exemplary steps of IHC can be summarized as follows: antigen retrieval (AR), addition of primary antibody, application of a secondary antibody that binds the primary antibody, and addition of a detection reagent to localize the primary antibody. The first step in IHC is typically antigen retrieval (AR), in which tissue is pretreated to retrieve antigens masked by fixation and make them more accessible to antibody binding. Antigen retrieval methods depend, in part, on the specific target antigen and antibody, but generally involve the breaking of protein cross-links caused by fixation, such as formalin, through chemical or physical means. Exemplary physical treatments include, but are not limited to, heat and ultrasound while exemplary chemical methods include, but are not limited to, enzyme digestion and denaturant treatment. One or both may be used to pretreat a single tissue.LBR 20276-P-US

[0063] The primary antibody can be monoclonal or polyclonal and is specific to a target antigen.The secondary antibody is targeted against the immunoglobulin of the species in which the primary antibody is produced. To visualize an antigen-antibody interaction, either the primary antibody or secondary antibody is labeled. In one aspect, the primary antibody is labeled and applied to the tissue. In a further aspect, the secondary antibody is labeled, allowing for signal amplification and use with more than one primary antibody. In one aspect, the label is an enzyme selected from horseradish peroxidase or alkaline phosphatase, which produce a colored product after incubation with a chromogenic substrate.

[0064] In one aspect, the disclosed comprise:performing, on a first tissue section, a first IHC assay for detection of Ki67, a second IHC assay for detection of CD3, and a third IHC assay for detection of CD20, wherein the IHC assays are performed in the following order: Ki67, CD3, and CD3, performing, on a second tissue section, a first IHC assay for detection of MUM1, a second IHC assay for detection of CD10, and a third IHC assay for detection of CD5, wherein the IHC assays are performed in the following order: MUM1, CD10, and CD5. and performing, on a third tissue section, a first IHC assay for detection of Bcl-2, a second IHC assay for detection of Cyclin D1, and a third IHC assay for detection of Bcl-6, wherein the IHC assays are performed in the following order: MUM1, Cyclin D1, and Bcl-6.

[0065] In one aspect, the IHC methods may comprise detecting the target antigens in layers, eachtissue sample being contacted with three different binding moieties, as follows:

[0066] A first layer in which Ki67 is detected, the detection comprising contacting the tissuesample with an anti-Ki67 binding moiety, for example, a primary monoclonal antibody specific for Ki67;

[0067] A second layer in which CD3 is detected, the detection comprising contacting the tissuesample with an anti-CD3 binding moiety, for example, a primary monoclonal antibody specific for CD3;LBR 20276-P-US

[0068] A third layer in which CD20 is detected, the detection comprising contacting the tissuesample with an anti-CD20 binding moiety, for example, a primary monoclonal antibody specific for CD20.

[0069] In a further aspect, each tissue sample is contacted with three different binding moieties,for example primary antibodies, as follows:

[0070] A first layer in which MUM1 is detected, the detection comprising contacting the tissuesample with an anti-MUM1 binding moiety, for example, a primary monoclonal antibody specific for MUM1;

[0071] A second layer in which CD10 is detected, the detection comprising contacting the tissuesample with an anti-CD10 binding moiety, for example, a primary monoclonal antibody specific for CD10;

[0072] A third layer in which CD5 is detected, the detection comprising contacting the tissuesample with an anti-CD5 binding moiety, for example, a primary monoclonal antibody specific for CD5.

[0073] In a further aspect, each tissue sample is contacted with three different binding moieties,for example primary antibodies, as follows:

[0074] A first layer in which Bcl-2 is detected, the detection comprising contacting the tissuesample with an anti-Bcl-2 binding moiety, for example, a primary monoclonal antibody specific for Bcl-2;

[0075] A second layer in which Cyclin D1 is detected, the detection comprising contacting thetissue sample with an anti-Cyclin D1 binding moiety, for example, a primary monoclonal antibody specific for Cyclin D1; and

[0076] A third layer in which Bcl-6 is detected, the detection comprising contacting the tissuesample with an anti-Bcl-6 binding moiety, for example, a primary monoclonal antibody specific for Bcl-6.LBR 20276-P-US

[0077] In accordance with IHC methods as described herein, a secondary antibody is employedwith each of the IHC assays described above. For example, in one aspect, each IHC assay as described above further includes contacting the layer with a secondary antibody specific to the species of the first antibody, the secondary antibody being conjugated to a chromogen. Chromogens

[0078] Chromogenic substrates have been used widely for immunohistochemistry for many yearsand for in situ hybridization. Typically, chromogenic substrates precipitate when activated by the appropriate enzyme. That is, in most instances, the chromogenic substance is converted from a soluble reagent into an insoluble, colored precipitate upon contacting the enzyme, and the resulting colored precipitate can be visualized. Upon enzymatic reaction with the target analyte, the chromagen undergoes a color change, the intensity of which is directly proportional to the concentration of the analyte present in the sample. By measuring the absorbance emitted by the chromogens at specific wavelengths, the presence, absence, and / or concentrations of multiple analytes can be determined simultaneously or sequentially.

[0079] The disclosed methods employ chromogens such that each layer of the sample asdescribed above comprises a brown chromogen, a red chromogen, and a green chromogen, the IHC assay forming a brown precipitate being performed on the sample first, the IHC assay forming a red precipitate being performed on the sample second, and the IHC assay forming a green precipitate being performed on the sample last. Applicant has found that, by selecting a target antigen based on where the target antigen is expressed in the cell, the selection and order of chromogens may be used to produce a readable visual display of the target antigens that allow for effective use of multiplex IHC to gather useful data for the characterization or diagnosis of NHL. Yet further, applicant has found that, with each of the three panels used, the use of the red and green chromogens, when used to detect each of the three target antigens as described, may create a fourth color – purple – when antigens are highly expressed within the tissues, which can further be used for diagnostic purposes. An example of a co-localization event forming the purple color is shown in FIG.9.

[0080] In one aspect, three IHC assays are performed per tissue sample, the three IHC assaysemploying three distinct chromogens (brown, red, and green). The three distinct chromogens usedLBR 20276-P-US in the disclosed methods allow for visualization of the presence and / or quantification of the concentration of at least three distinct targets in a tissue sample. Brown Chromogen

[0081] In one aspect, the disclosed methods utilize a first chromogen, the first chromogenproducing a brown signal. The first IHC assay performed on a tissue section may comprise contacting the tissue section with a primary antibody (for example, a mouse or rabbit monoclonal antibody), wherein the primary antibody is specific to target molecule selected from Ki67 (the antibody being referred to as anti-Ki67), MUM1 (the antibody being referred to as anti-MUM1), or Bcl-2 (the antibody being referred to as anti-Bcl-2). Following a wash step, a secondary antibody specific for the primary antibody is then applied to the tissue section for a second incubation period. In one aspect, the secondary antibody is an anti-species antibody such as anti- mouse or anti-rabbit. In one aspect, the secondary antibody may be conjugated to an enzyme that is capable of precipitating a first chromogen. In this aspect, the first chromogen is a brown chromogen, which is applied to the tissue section. In the presence of the target molecule, the brown chromogen is precipitated as a result of contact with the enzyme conjugated with the secondary prior to the secondary antibody for further amplification of the signal produced by the chromogen, as will be readily understood by one of ordinary skill in the art.

[0082] Brown chromogens suitable for use with the disclosed methods will be understood by oneof ordinary skill in the art. For example, in one aspect, the brown chromogen is DAB, or 3,3'- diaminobenzidine. In this aspect, the enzyme used is HRP, which catalyzes the oxidation of DAB in the presence of hydrogen peroxide. This reaction results in the formation of a brown-colored insoluble precipitate at the site of enzyme activity. Upon visualization, the presence of the antigen (Ki67, MUM1, and / or Bcl-2) is indicated by brown staining at the location of the target protein. Red Chromogen

[0083] In one aspect, the disclosed methods utilize a second chromogen, the second chromogenproducing a red signal. In general, the IHC assay using the second chromogen is carried out following the IHC assay using the first chromogen; i.e., the detection of Ki67, MUM1, or Bcl-2 isLBR 20276-P-US carried out first using a brown chromogen as described above prior to the second IHC assay which uses a red chromogen. The second IHC assay comprises contacting the tissue section with a primary antibody (for example, a mouse or rabbit monoclonal antibody) specific to a target molecule selected from CD3 (the antibody being referred to as anti-CD3), CD10 (the antibody being referred to as anti-CD10), or Cyclin D1 (the antibody being referred to as anti-Cyclin D1), and the primary antibody is incubated with the tissue section for a first incubation period. Following a wash step, a secondary antibody is then applied to the tissue section for a second incubation period. In one aspect, the secondary antibody is an anti-species antibody such as anti- mouse or anti-rabbit. In one aspect, the secondary antibody may be conjugated to an enzyme that is capable of precipitating a second chromogen. In this aspect, the second chromogen is a red chromogen, which is applied to the tissue section. In the presence of the target molecule, the red chromogen is precipitated as a result of contact with the enzyme conjugated with the secondary secondary antibody for further amplification of the signal produced by the chromogen.

[0084] Red chromogens suitable for use with the disclosed methods will be understood by one ofordinary skill in the art. For example, in one aspect, the red chromogen may be Permanent Red (also known as Fast Red), Nathol AS-MX Phosphate, Bromo-Chloro-Indolyl Phosphate (BCIP) with Nitroblue Tetrazolium (NBT), New Fuchin, Vector Red, or combinations thereof. In this aspect, the enzyme is alkaline phosphatase (AP), which causes the formation of a red-colored insoluble precipitate at the site of enzyme activity. Upon visualization, the presence of the antigen (CD3, CD10, or Cyclin D1) is indicated by red staining at the location of the target protein. Green Chromogen

[0085] In one aspect, the disclosed methods utilize a third chromogen, the third chromogenproducing a green signal. In general, the IHC assay using the third chromogen is carried out following the second IHC assay using the second chromogen. The third IHC assay performed on a tissue section may comprise contacting the tissue section with a primary antibody (for example, a mouse or rabbit monoclonal antibody), wherein the primary antibody is specific to target molecule selected from CD20 (the antibody being referred to as anti-CD20), CD5 (the antibody being referred to as anti-CD5), or Bcl-6 (the antibody being referred to as anti-Bcl-6). FollowingLBR 20276-P-US a wash step, a secondary antibody is then applied to the tissue section for a second incubation period. In one aspect, the secondary antibody is an anti-species antibody such as anti-mouse or anti-rabbit. In one aspect, the secondary antibody may be conjugated to an enzyme that is capable of precipitating a second chromogen. In this aspect, the third chromogen is a green chromogen, which is applied to the tissue section. In the presence of the target molecule, the green chromogen is precipitated as a result of contact with the enzyme conjugated with the secondary antibody.

[0086] Green chromogens suitable for use with the disclosed methods will be understood by oneof ordinary skill in the art. For example, in one aspect, the green chromogen may be 4-Chloro-1- Naphthol (4-CN), Leucocrystal Violet, Fast Green FCF, Malachite Green, Rhodamine Green carboxylic acid succinimidyl ester (DY-505),], or combinations thereof. In this aspect, the enzyme may be HRP, which causes the formation of a green-colored insoluble precipitate at the site of enzyme activity. Upon visualization, the presence of the antigen (CD20, CD5, or Bcl-6) is indicated by green staining at the location of the target protein. Counterstain

[0087] The disclosed methods may further comprise a cytochemical staining procedure forfurther visualization of cells or cell compartments such that their structures can be more readily visualized under a microscope, such staining procedure being carried out in Post-Processing step (140). For example, a counterstain may be used prior to cover-slipping to render the immunohistochemical stain more distinct. Such staining procedures are known to those of skill in the art and may comprise, for example, staining for acidophilic or basophilic structures, of subcellular regions (e.g. the nucleus, the mitochondria, the golgi, the cytoplasm etc.), of specific molecules (of chromosomes, of lipids, of glycoproteins, of polysaccharids etc.) in the cytological specimens. Fluorescence dyes such as DAPI, Quinacrin, Chromomycin, etc. may be employed. Furthermore, chromogenic dyes such as Azan, Acridin-orange, Hematoxylin, Eosin, Sudan-red, Thiazin-stains (Toluidin-blue, Thionin) may be applied. In other aspects, staining procedures such as Pap-staining, Giemsa-staining, Hematoxylin-Eosin staining, van-Gieson staining, Schiff- staining (using Schiff reagent), staining procedures employing precipitation of metals (such as e.g. of silver in staining procedures employing Silver Nitrate) or insoluble stains such as e.g. of Turnbulls-blue (or other insoluble metal cyanides), etc. may be used.LBR 20276-P-US III. Example Slide-Staining Instrument

[0088] In one aspect, disclosed are methods for the preparation of samples for IHC detectionmethods which may employ, in whole or in part, the slide-staining instrument (10) described herein. It should be understood that other instruments may be used to carry out the disclosed methods, for example that described in U.S. Patent 11493411, U.S. Patent Publication No. 2021 / 0293670, and U.S. Patent Publication No. 2023 / 0022299. FIG. 1 shows an example of a slide-staining instrument (10) that is generally configured to automatically stain tissue mounted on microscope slides. Slide-staining instrument (10) includes a group fluid dispenser (12) (also referred to as an aspirating probe) mounted to a robotic arm (14) and a plurality of slide staining assemblies (40) disposed beneath robotic arm (14). As will be described in greater detail below, robotic arm (14) and slide staining assemblies (40) are generally configured to operate cooperatively to automatically dispense reagents, for example, IHC assay reagents, stain, probes, enzymes, and the like, onto one or more microscope slides disposed within each slide staining assembly (40).

[0089] To supply reagents, slide-staining instrument (10) includes a bulk container rack (20) anda reagent platform (30). Bulk container rack (20) is configured to hold a plurality of bulk containers (22). Bulk containers (22) may be configured to contain a relatively large quantity of reagent that may be communicated to group fluid dispenser (12) and / or each slide staining assembly (40). Examples of reagents that may be stored in bulk containers (22) may include, for example, formalin solutions, wash solutions, deionized water, buffered solutions such as phosphate buffered saline (PBS), and / or alcohol, and additional reagents useful to carrying out the disclosed methods. Additionally, one or more of bulk containers (22) may be configured to contain waste fluids communicated from group fluid dispenser (12) and / or each slide staining assembly (40). In some examples, such waste fluids may be separated by slide-staining instrument (10) into bulk waste and hazardous waste. Thus, separate bulk containers (22) may be included for bulk waste and hazardous waste. A plurality of syringe pumps (24) may be included proximate bulk containers (22) to communicate fluid to or from bulk containers (22) relative to other portions of slide-staining instrument (10).LBR 20276-P-US

[0090] Reagent platform (30) is generally configured to receive one or more reagent trays (32).Each reagent tray (32) is configured to hold a plurality of reagent containers (34). Reagent containers (34) may be configured to contain a relatively small quantity of reagent for communication to group fluid dispenser (12) and / or slide staining assembly (40). Each reagent tray (32) may include a particular predetermined combination of reagent containers (34) to form a predefined reagent system. Such predefined reagent systems may be ready-to-use and are discarded upon exhaustion during use with a particular protocol. Each reagent tray (32) is registered upon insertion into reagent platform (30). Although not shown, it should be understood that group fluid dispenser (12) may access reagent containers (34) directly. Also, one or more syringe pumps similar to syringe pumps (24) described above may be included to communicate reagents from reagent containers (34) to group fluid dispenser (12), slide staining assembly (40), and / or other portions of slide-staining instrument (10).

[0091] FIG. 2 shows an example of a slide tray (60) that may be used with slide-staininginstrument (10) to hold one or more slides during the application of reagents via slide-staining instrument (10). Slide tray (60) includes a plurality of slide recesses (62) separated by rails (64). Each slide recess (62) is generally configured to receive an individual slide. Although not shown, it should be understood that each slide recess (62) may include an open bottom in some examples such that each slide may be supported within slide tray (60) at the periphery of each slide rather than in a center or middle of each slide. Thus, in some examples, each slide recess (62) may include one or more ledges, protrusions, supports or other features configured to support the outer edges of a slide received therein.

[0092] Slide tray (60) further includes a plurality of couplers (66) with one or more couplers (66)being associated with a corresponding slide recess (62). Couplers (66) are generally configured to removably couple to a cover (70) (also referred to as a covertile). As will be described in greater detail below, covers (70) may be used to secure a slide in position on or within each slide recess (62). In some examples, couplers (66) may include one or more recesses on one side of a counterpart slide recess (62) and one or more snap-fits on another side of a counterpart slide recess (62). Of course, in other examples, couplers (66) may have a variety of other configurations as will be apparent to those of ordinary skill in the art in view of the teachings herein.LBR 20276-P-US

[0093] FIG. 3 shows the assembly of slide tray (60) and cover (70) to hold a slide (80) in greaterdetail. As can be seen, slide (80) may be disposed between cover (70) and slide tray (60) within a given slide recess (62). Cover (70) is generally configured to hold slide (80) in position and rest over slide (80) during staining. In this configuration, capillary action may draw reagent dispensed to slide (80) between cover (70) and slide (80). Such capillary action may be desirable to provide gentle and uniform tissue coverage with the dispensed reagent, thereby minimizing the volume of reagent used and protecting tissue from drying between applications. In some examples, cover (70) may be reusable, while cover (70) may be disposable in other examples.

[0094] FIG. 4 shows an example of slide staining assembly (40) in greater detail. As describedabove, slide-staining instrument includes a plurality of slide staining assemblies (40) generally configured to operate cooperatively with robotic arm (14) to automatically dispense reagents onto one or more microscope slides disposed within each slide staining assembly (40). Specifically, each slide staining assembly (40) is configured to receive a corresponding slide tray (60). When slide tray (60) is loaded with slides such as slide (80) robotic arm (14) along with slide staining assembly (40) itself may be used to dispense reagents onto each slide loaded on slide tray (60).

[0095] Slide staining assembly (40) includes a top cover (42), a guide rail (44), and a batch fluiddispenser (46). Slide staining assembly (40) is configured removably receive and support slide tray (60) beneath top cover (42). Batch fluid dispenser (46) is configured to move along guide rail (44) relative to slide tray (60) to dispense slide reagents and buffers from one or more of bulk containers (22) onto slides contained within slide tray (60). In some examples, batch fluid dispenser may alternatively be characterized as a fluid dispensing robot. Although not shown, it should be understood that slide staining assembly (40) may include a plurality of heating elements corresponding to each slide recess (62) of slide tray (60). A portion of such heating elements can be incorporated into a support for each slide, which can be configured to communicate heat from heating elements to each slide. Thus, slide staining assembly (40) is configured to maintain each slide disposed within slide tray (60) at a selected temperature independently of the other slides.

[0096] In use, robotic arm (14) is used to position group fluid dispenser (12) relative to slidestaining assembly (40) and reagent platform (30). Robotic arm (14) may then be moved to reagent platform (30) to gather one or more reagents with group fluid dispenser (12). Optionally, one orLBR 20276-P-US more reagents may be mixed or otherwise manipulated in a separate area of slide-staining instrument (10). Robotic arm (14) may then move to a selected slide staining assembly (40) to dispense gathered reagents onto one or more slides disposed within the selected slide staining assembly (40).

[0097] Simultaneously with use of robotic arm (14) to dispense reagents, or separately therefrom,each slide staining assembly (40) may dispense reagents onto one or more slides disposed within a respective slide staining assembly (40). Specifically, batch fluid dispenser (46) may move along guide rail (44) to communicate one or more reagents from bulk containers (22) onto slides via syringe pumps (24) and other components associated with slide-staining instrument (10). Thus, robotic arm (14) and slide staining assemblies (40) are configured to operate cooperatively to efficiently complete staining protocols with one or more steps of a given staining protocol being performed simultaneously with one or more other steps. During the performance of such staining protocols, heaters included within each slide staining assembly (40) are used to maintain the temperature of individual slides at a selected temperature in accordance with such staining protocols. IV. Immunohistochemistry Assay (200)

[0098] As described above, slide-staining instrument (10) may be configured to perform a varietyof protocols where robotic arm (14) and / or slide staining assemblies (40) may automatically apply a variety of reagents to slides under various predetermined parameters. Such slide staining protocols may include a series of steps where various reagents may be applied to individual slides in a predetermined order, in a predetermined volume at predetermined temperatures for predetermined periods of time.

[0099] FIG. 5 depicts an exemplary Immunohistochemistry Assay Protocol (100). For example,a tissue section is prepared from a tissue sample obtained from an individual as described above. In one aspect, the tissue sample is a FFPE tissue section which is placed on a slide and deparaffinized using the BOND Dewax protocol, which removes paraffin wax from the tissue section before rehydration and staining. In the first phase of the Immunohistochemistry Assay Protocol (100), Layer 1 Staining Protocol (110), which comprises a first IHC assay, is carried out for detection of a first target antigen. Immunohistochemistry Assay Protocol (100) begins withLBR 20276-P-US incubation of the tissue section with an epitope retrieval solution, which assists in exposing the desired antigen. Exemplary epitope retrieval solutions are known in the art. An exemplary epitope retrieval solution comprises EDTA, Tris Base, Sodium Citrate, Proteinase K, Trypsin, and Pepsin. In aspects, the epitope retrieval solution is BOND Epitope Retrieval Solution 1, a ready to use, citrate-based pH 6 epitope retrieval solution for the heat-induced epitope retrieval (HIER) of formalin-fixed, paraffin-embedded tissue on the BOND automated system, which restores epitopes that have been modified by formalin fixation, allowing accessibility of the primary antibody to the epitope. The epitope retrieval solution incubation step (111) may be carried out at a temperature sufficient to expose the antigens of interest and may be carried out for a period of time ranging from about 30 seconds to about 5 minutes, or from about 1 minute to about 10 minutes, or about 2 minutes. The duration and temperature used for incubation of the reagents during the epitope retrieval solution incubation step (111) may be predetermined and programmed into the slide- staining instrument (10) and may depend on the specific epitope retrieval solution used. Following epitope retrieval solution incubation step (111), the tissue section is subjected to a hydrogen peroxide treatment (112) to quench endogenous peroxidase activity. The hydrogen peroxide treatment step (112) may employ, for example, a solution of about 3% to 4% hydrogen peroxide solution applied to the tissue section for about 30 seconds to about 5 minutes, or from about 1 minute to about 10 minutes, or about 2 minutes. Primary antibody (113) is then applied to the tissue section and incubated for a time and temperature sufficient to allow specific binding to the target. For example, the primary antibody may be incubated with the tissue section for an incubation period of about 1 to about 30 minutes or about 5 to about 25 minutes, or about 10 to about 20 minutes, or about 15 minutes. The tissue section is then subjected to post-primary linker minutes to about 30 minutes, or about 10 minutes to about 20 minutes, or about 15 minutes followed by contact with a secondary antibody conjugated to an enzyme (115). In one aspect, the enzyme used in the Layer 1 Staining Protocol (110) is horseradish peroxidase. In Layer 1 Staining Protocol (110), a brown chromogen application step (116) is carried out. Exemplary brown Diaminobenzidine as used in BOND Polymer Refine Detection available from Leica Biosystems, Product Code DS9800. Brown chromogen application step (116) is carried out for a time and temperature sufficient to allow precipitation of the chromogen, for example, for about 5 minutesLBR 20276-P-US to about 20 minutes, or about 7 minutes to about 15 minutes, or about 10 minutes. Elution (117) is then carried out, in which a neutralization wash (e.g. EDTA based pH 9 solution) is applied to bring the tissue to neutral pH. In one aspect, elution (180) may be carried out at a temperature of about 15 minutes, or about 10 minutes. In Layer 1 Staining Protocol (110), the primary antibody may be selected from anti-Ki67, anti-MUM1, or anti-Bcl-2, as described herein.

[0100] In the second phase of the Immunohistochemistry Assay Protocol (100), Layer 2 StainingProtocol (120) , which comprises a second IHC assay, is carried out for detection of a second target antigen. Following elution (117), Layer 2 Staining Protocol (120) is carried out on the tissue section Layer 2 Staining Protocol (120) is also shown in FIG. 5. The tissue section of Layer 1 Staining Protocol (110) described above is incubated with primary antibody (121) for a time and temperature sufficient to allow specific binding to the target. In Layer 2 Staining Protocol (120), the primary antibody is selected from anti-CD3, anti-CD10, and anti-Cyclin D1. The tissue section is then subjected to post-primary linker reagent (122), followed by contact with a secondary antibody (123), which is conjugated to the enzyme alkaline phosphatase (AP). In Layer 2 Staining Protocol (120), the chromogen precipitated by the enzyme is a red chromogen (for example that used in BOND Polymer Refine Red Detection, an IVD labeled red detection system, available from Leica Biosystems, Product Code DS9390). Red chromogen application step (124) is carried out for a time and temperature sufficient to allow precipitation of the red chromogen, for example, for about 5 minutes to about 20 minutes, or about 7 minutes to about 15 minutes, or about 10 minutes. Elution (125) is carried out as described above.

[0101] In the third phase of the Immunohistochemistry Assay Protocol (100), Layer 3 StainingProtocol (130), which comprises a third IHC assay, is carried out for detection of a third target antigen. Following elution (125), Layer 3 Staining Protocol (130) is carried out on the tissue section. Layer 3 Staining Protocol (130) is also shown in FIG. 5. The tissue section of Layer 1 Staining Protocol (110) and Layer 2 Staining Protocol Staining Protocol (120) above is incubated with primary antibody (131) for a time and temperature sufficient to allow specific binding to the target, as described above. In Layer 3 Staining Protocol (130), the primary antibody is selected from anti-CD20, anti-CD5, and anti-Bcl-6. The tissue section is then subjected to post-primary linker reagent (132), followed by contact with a secondary antibody conjugated to an enzymeLBR 20276-P-US (133). In one aspect, the enzyme used is horseradish peroxidase. In Layer 3 Staining Protocol (130), a green chromogen is applied. Green chromogen application step (134) is carried out for a time and temperature sufficient to allow precipitation of the green chromogen, for example, for about 5 minutes to about 20 minutes, or about 7 minutes to about 15 minutes, or about 10 minutes.

[0102] The tissue section is then counterstained with a suitable counterstain in post-processingstep (140), for example, hematoxylin solution, and washed to remove excess reagent. The resulting tissue section may then be visualized for the presence of brown, red, green, and, where co- localization occurs, purple (the purple arising from overlap of red and green chromogens). An example of a co-localization event forming the purple color is shown in FIG.9.

[0103] It should be understood that the above protocols may include one or more washing stepsbetween any one or more of the steps or protocols described above prior to the dehydration step with increased concentration of alcohol ranging from 70% to 100%. Such wash steps may be performed with a variety of solutions such as deionized water, phosphate buffered saline, or other buffered solutions, or various combinations thereof.

[0104] Incubation times of any of the described steps may vary. For example, any dispense cyclein which a reagent is applied to the sample may include an incubation. Incubations are generally performed with the given slide being heated to a predetermined temperature. The incubation time may be varied depending on the desired outcome and the particular reagents (for example, the particular probe) that is used. For example, the incubation time may be about 20 minutes for a dispense cycle, or about 30 minutes for a dispense cycle, or about 60 minutes for a dispense cycle. Although particular incubation times are described, it should be understood that in other examples, either a different minimum incubation time or a different maximum incubation time may be used. Control System (310)

[0105] FIG. 6 shows an example of a control system (310) that may be used to implement theImmunohistochemistry Assay Protocol (100) as described herein using slide-staining instrument (10). Control system (310) includes a processor (320) in communication with a first memory device (322) for storing computer program code and a second memory device (324) for storing data generated by processor (320) when implementing the computer program code, viaLBR 20276-P-US communications infrastructure (326). Optionally, the functions of first memory device (322) and second memory device (324) may be combined in a single memory device or a plurality of memory devices in communication with each other.

[0106] Control system (310) further includes one or more inputs and one or more outputs tofacilitate operator interaction with control system (310). For instance, in the present example, control system (310) includes a display interface (328) and corresponding display (330) to enable operator interaction with control system (310). In some examples, display (330) may be configured as a touch screen display so display (330) may both receive operator input and communicate operator outputs. In addition, or in the alternative, control system (310) may include one or more operator input features such as a keyboard or keypad to provide a dedicated operator input feature.

[0107] Control system (310) also includes driver modules (334, 336, 338, 340, 342) forcontrolling the motors, pumps, scanners, heaters and other devices (346, 348, 350, 352, 354) used for operation of the slide-staining instrument (10). It will be appreciated that in FIG.6, examples only of driver modules and devices are depicted, and a person of skill in the art will be able to determine the driver modules and devices required to implement slide-staining instrument (10) to provide the functionality described herein.

[0108] It should be understood that memory devices (322, 324) may be disposed within a portionof slide-staining instrument (10) or may be hosted remote from slide-staining instrument (10) in data communication with processor (320). Regardless of the specific configuration, the processor (320) is configured to read instructions from first memory device (322) to operate the slide-staining instrument (10) to complete one or more slide-staining protocols. Such staining protocols (e.g. order of reagents to be dispensed by batch fluid dispenser (46) and group fluid dispenser (12) to slides) are stored in a protocol database (356) accessible by processor (320) via communications infrastructure (326), such that processor (320) can configure batch fluid dispenser (46) and group fluid dispenser (12) to dispense reagents to slides disposed within each slide staining assembly (40) in a predetermined order and at predetermined intervals.LBR 20276-P-US Examples

[0109] The following non-limiting examples are provided to further illustrate embodiments ofthe invention disclosed herein. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches that have been found to function well in the practice of the invention, and thus may be considered to constitute examples of modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes may be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. Example I.

[0110] mounted onto charged slides. Slides were baked at 60°C for 30 minutes. All steps there on forth were completed on the BOND-Rx automated staining instrument (software version 7) utilizing the sequential staining method. All steps unless otherwise stated are completed at ambient temperature.

[0111] Layer 1

[0112] Slides are deparaffinized using the BOND Dewax protocol and heat induced epitoperetrieved using BOND Epitope Retrieval Solution 2 for 20 mins at 100oC. The specimen is incubated with hydrogen peroxide for 5 minutes to quench endogenous peroxidase activity and a primary antibody (BOND Ready-to-Use reagent) of monoclonal mouse or rabbit origin is applied chromogen is applied to the tissue for 10 minutes, and produces a brown precipitate, localized to the cellular compartment of the epitope in detection.

[0113] An antibody elution step is applied to the tissue before moving onto the next sequentialstaining round. This elution method includes a heated temperature of 50oC for 10 minutes, followed by a neutralization wash to ensure the tissue is brought back to a neutral pH.LBR 20276-P-US

[0114] Layer 2

[0115] The specimen is incubated with a primary antibody (BOND Ready-to-Use reagent) ofmonoclonal mouse or rabbit origin is applied following an incubation of 15 minutes. A secondary phosphatase for 30 minutes. The Refine red chromogen is applied to the tissue (10+5 minutes) and produces a red precipitate localized to the cellular compartment of the epitope in detection.

[0116] An antibody elution step is applied to the tissue before moving onto the next sequentialstaining round. This elution method includes a heated temperature of 50oC for 10 minutes, followed by a neutralization wash to ensure the tissue is brought back to a neutral pH.

[0117] Layer 3

[0118] Slides are incubated with BOND Epitope Retrieval Solution 2 for 5 minutes. Thespecimen is incubated with a primary antibody (BOND Ready-to-Use reagent) of monoclonal mouse or rabbit origin is applied following an incubation of fifteen minutes. A secondary reagent for 10 minutes. The green chromogen is applied to the tissue (10 minutes) and produces a green precipitate localized to the cellular compartment of the epitope in detection.

[0119] Final protocol steps include a counterstain dispense of Hematoxylin solution for 1 minuteat ambient temperature, followed by the application of BOND Wash Solution to allow blueing of the reagent to take place. LBS Product Code Antibody / Clone Multiplex Panel Detection LayerChromogenic DetectionLBR 20276-P-US PA0046 CYCLIN D1 (EP12) 2 Refine Red PA0204 3 Green

[0120] FIGS. 7A-7C depict an exemplary result following the assay as described in Example 1.FIG. 7A depicts Ki67 / CD3 / CD20 staining on Follicular Lymphoma positive tissue. FIG. 7B depicts Ki67 / CD3 / CD20 staining on Mantle Cell Lymphoma positive tissue. FIG. 7C depicts Ki67 / CD3 / CD20 staining on Diffuse Large B Cell Lymphoma positive tissue. For each of FIGS. 7A-7C, the presence of Ki67 is indicated with brown chromogen, the presence of CD3 is indicated with red chromogen, and the presence of CD20 is indicated with green chromogen.

[0121] FIGS. 8A-8C depict an exemplary result following the assay as described in Example 1.FIG. 8A depicts MUM1 / CD10 / CD5 staining on Follicular Lymphoma positive tissue. FIG. 8B depicts MUM1 / CD10 / CD5 staining on Mantle Cell Lymphoma positive tissue. FIG. 8C depicts MUM1 / CD10 / CD5 staining on Diffuse Large B Cell Lymphoma positive tissue. For each of FIGS. 8A-8C, the presence of MUM1 is indicated with brown chromogen, the presence of CD10 is indicated with red chromogen, and the presence of CD5 is indicated with green chromogen.

[0122] FIG 9 illustrates co-localization events (circled) in which CD10 (labeled with redchromogen) and CD5 (labeled with green chromogen) form a fourth color (purple) in a small subset of B cells, following the methods described in Example 1.

[0123] FIGS. 10A-10C depict an exemplary result following the assay as described in Example1. FIG.10A depicts Bcl-2 / Cyclin D1 / Bcl-6 staining on Follicular Lymphoma positive tissue. FIG. 10B depicts Bcl-2 / Cyclin D1 / Bcl-6 staining on Mantle Cell Lymphoma positive tissue. FIG.10C depicts Bcl-2 / Cyclin D1 / Bcl-6 staining on Diffuse Large B Cell Lymphoma positive tissue. For each of FIGS.10A-10C, the presence of Bcl-2 is indicated with brown chromogen, the presence of Cyclin D1 is indicated with red chromogen, and the presence of Bcl-6 is indicated with green chromogen. IV. Exemplary Combinations

[0124] The following examples relate to various non-exhaustive ways in which the teachingsherein may be combined or applied. It should be understood that the following examples are notLBR 20276-P-US intended to restrict the coverage of any claims that may be presented at any time in this application or in subsequent filings of this application. No disclaimer is intended. The following examples are being provided for nothing more than merely illustrative purposes. It is contemplated that the various teachings herein may be arranged and applied in numerous other ways. It is also contemplated that some variations may omit certain features referred to in the below examples. Therefore, none of the aspects or features referred to below should be deemed critical unless otherwise explicitly indicated as such at a later date by the inventors or by a successor in interest to the inventors. If any claims are presented in this application or in subsequent filings related to this application that include additional features beyond those referred to below, those additional features shall not be presumed to have been added for any reason relating to patentability.

[0125] Exemplary combinations

[0126] Example 1

[0127] A tissue staining method for characterization of non-Hodgkin’s lymphoma comprising:performing, on a first tissue section, a plurality of immunohistochemistry (IHC) assays comprising a first IHC assay for detection of Ki67, said first IHC assay comprising a first chromogen; a second IHC assay for detection of CD3, said second IHC assay comprising a second chromogen; a third IHC assay for detection of CD20, said third IHC assay comprising a third chromogen; performing, on a second tissue section, a plurality of immunohistochemistry assays comprising a first IHC assay for detection of MUM1, said first IHC assay comprising a first chromogen; a second IHC assay for detection of CD10, said second IHC assay comprising a second chromogen; a third IHC assay for detection of CD5, said third IHC assay comprising a third chromogen; performing, on a third tissue section, an immunohistochemistry assay, said immunohistochemistry assay comprising a first IHC assay for detection of Bcl-2, said first IHC assay comprising a first chromogen; a second IHC assay for detection of CyclinD1, said second IHC assay comprising a second chromogen; and a third IHC assay for detection of Bcl-6, said third IHC assay comprising a third chromogen.

[0128] Example 2LBR 20276-P-US

[0129] The method of any preceding example, each said first assay being performed on said firsttissue section prior to said second assay, and each said second assay being performed prior to each said third assay.

[0130] Example 3

[0131] The method of any preceding example, said first IHC assay for detection of Ki67comprising contacting said first tissue section with a primary antibody having specific binding to Ki67.

[0132] Example 4

[0133] The method of any preceding example, said second IHC assay for detection of CD3comprising contacting said first tissue section with a primary antibody having specific binding to CD3.

[0134] Example 5

[0135] The method of any preceding example, said third IHC assay for detection of CD20comprising contacting said first tissue section with a primary antibody having specific binding to CD20.

[0136] Example 6

[0137] The method of any preceding example, said first IHC assay for detection of MUM1comprising contacting said first tissue section with a primary antibody having specific binding to MUM1.

[0138] Example 7

[0139] The method of any preceding example, said second IHC assay for detection of CD10comprising contacting said first tissue section with a primary antibody having specific binding to CD10.

[0140] Example 8LBR 20276-P-US

[0141] The method of any preceding example, said third IHC assay for detection of CD5comprising contacting said first tissue section with a primary antibody having specific binding to CD5.

[0142] Example 9

[0143] The method of any preceding example, said first IHC assay for detection of BCL2comprising contacting said first tissue section with a primary antibody having specific binding to Bcl-2.

[0144] Example 10

[0145] The method of any preceding example, said second IHC assay for detection of CyclinD1comprising contacting said first tissue section with a primary antibody having specific binding to CyclinD1.

[0146] Example 11

[0147] The method of any preceding example, said third IHC assay for detection of Bcl-6comprising contacting said first tissue section with a primary antibody having specific binding to Bcl-6.

[0148] Example 12

[0149] The method as in any of examples 3-11, said primary antibody being a monoclonalantibody.

[0150] Example 13

[0151] The method as in any of examples 3-12, said primary antibody being a mouse or rabbitmonoclonal antibody.

[0152] Example 14

[0153] The method as in any of examples 3-13, further comprising an anti-species linker, saidanti-species linker being an antibody which specifically binds to said primary antibody.LBR 20276-P-US

[0154] Example 15

[0155] The method as in any of examples 1-14, further comprising a secondary antibody.

[0156] Example 16

[0157] The method as in example 15, said secondary antibody being an anti-species monoclonalantibody.

[0158] Example 17

[0159] The method as in any of examples 14-15, said secondary antibody being an anti-speciesmonoclonal antibody specific to said primary antibody.

[0160] Example 18

[0161] The method as in any of examples 14-16, said secondary antibody being an anti-speciesmonoclonal antibody specific to said anti-species linker.

[0162] Example 19

[0163] The method as in any of examples 16-18, said anti-species monoclonal antibodycomprising an enzyme for precipitating a chromogen.

[0164] Example 20

[0165] The method of example 19, said enzyme for precipitating a chromogen being selectedfrom alkaline phosphatase (AP) and / or horseradish peroxidase (HRP).

[0166] Example 21

[0167] The method of any of examples 19-20, said first chromogen, said second chromogen, andsaid third chromogen reacting with said enzyme to form a precipitated product.

[0168] Example 22LBR 20276-P-US

[0169] The method of example 21, said precipitated product binding to tissues adjacent to wherethe primary antibody is bound.

[0170] Example 23

[0171] The method as in any of examples 1-22, said first chromogen being a brown chromogen.

[0172] Example 24

[0173] The method as in any of examples 1-23, said second chromogen being a red chromogen.

[0174] Example 25

[0175] The method as in any of examples 1-24, said third chromogen being a green chromogen.

[0176] Example 26

[0177] The method of example 1 wherein said first, second, and third IHC assay comprisecontacting said tissue section with a primary antibody; contacting said tissue sample with a secondary antibody conjugated to an enzyme configured to catalyze said chromogen, said secondary antibody being specific to said primary antibody; and detecting said chromogen.

[0178] Example 27

[0179] The method of example 1 or 2, each said tissue section comprising tissue obtained froman individual having, or suspected of having, non-Hodgkin’s lymphoma.

[0180] Example 28

[0181] The method of any preceding example, each said tissue section comprising tissue obtainedfrom the same individual.

[0182] Example 29

[0183] The method of any preceding example, each said tissue section comprising tissue fromthe same biopsy sample.LBR 20276-P-US

[0184] Example 30

[0185] The method as in any of examples 1-29, each said tissue section comprising tissue fromdifferent biopsy sample.

[0186] Example 31

[0187] The method as in any of examples 1-30, further comprising staining said first, second,and / or third tissue section with hematoxylin.

[0188] Example 32

[0189] The method as in any of examples 1-31, further comprising imaging said first, second,and / or third tissue section.

[0190] Example 33

[0191] The method of any preceding example, further comprising detecting said first chromogen,said second chromogen, and said third chromogen.

[0192] Example 34

[0193] The method as in any of examples 1-33, wherein said first chromogen, said secondchromogen, and said third chromogen are simultaneously detected.

[0194] Example 35

[0195] An automated slide staining apparatus for assaying one or more tissue samples disposedon a slide, the apparatus comprising: a controller; a slide staining assembly, the slide staining assembly being configured to receive one or more of the slides; and at least one fluid dispensing robot configured by the controller to dispense a plurality of reagents to the one or more slides received in the slide staining assembly to treat the one or more tissue samples respectively, the at least one fluid dispensing robot being configured by the controller to dispense one or more reagents of the plurality of reagents in a predetermined sequence corresponding to an IHC protocol for the one or more slides received in the slide staining assembly to treat the one or more tissue samples disposed on each slide independently, the at least one fluid dispensing robot being furtherLBR 20276-P-US configured by the controller to dispense a reagent for specific binding of Ki67, MUM1, or Bcl-2, and a first chromogen, a reagent for specific binding of CD3, CD10, or Cyclin D1 and a second chromogen, and a reagent for specific binding of CD20, CD5, and Bcl-6 and a third chromogen; in a predetermined sequence for performing a plurality of IHC staining protocols for the one or more slides received in the slide staining assembly to treat the one or more tissue samples disposed on each slide independently, and the at least one fluid dispensing robot being further configured by the controller to perform the predetermined sequence corresponding to the IHC staining protocol.

[0196] Example 36

[0197] The apparatus example 35, the slide staining assembly being configured by the controllerto heat each slide received by the slide staining assembly independently to an incubation temperature.

[0198] Example 37

[0199] The apparatus as in any of examples 35-36, the slide staining assembly being configuredby the controller to heat each slide received by the slide staining assembly independently to an incubation temperature during the staining protocol.

[0200] Example 38

[0201] A method for an automated multiplex IHC assay of one or more tissue samples disposedon a slide, the method comprising dispensing one or more reagents to the one or more tissue samples using one or more automated fluid dispensers in a series of staining steps associated with an IHC protocol, said reagents comprising a reagent for specific binding of Ki67, MUM1, or Bcl- 2, and a first chromogen, a reagent for specific binding of CD3, CD10, or Cyclin D1 and a second chromogen, and a reagent for specific binding of CD20, CD5, and Bcl-6 and a third chromogen;.

[0202] Example 39

[0203] The method of example 38, further comprising dispensing one or more reagents to the oneor more tissue sections using one or more automated fluid dispensers in a series of sample preparation steps associated with a sample preparation protocol.LBR 20276-P-US

[0204] Example 40

[0205] The method of any of examples 38-39, the one or more reagents comprising a primaryantibody specific to Ki67, a primary antibody specific to MUM1, a primary antibody specific to Bcl-2, a primary antibody specific to CD3, a primary antibody specific to CD10, a primary antibody specific to Cyclin D1 a primary antibody specific to CD20, a primary antibody specific to CD5, and a primary antibody specific to Bcl-6.

[0206] Example 41

[0207] The method of example 40, the primary antibody comprising a mouse monoclonalantibody.

[0208] Example 42

[0209] The method of any of examples 40-41, the primary antibody comprising a rabbitmonoclonal antibody.

[0210] Example 43

[0211] The method of any of examples 39-42, the one or more reagents comprising a secondaryantibody specific to a primary antibody of example 40.

[0212] Example 44

[0213] The method of example 43, the secondary antibody comprising an anti-species antibody.

[0214] Example 45

[0215] The method of any of examples 43-44, the secondary antibody comprising an anti-mouseand / or anti-rabbit antibody.

[0216] Example 46

[0217] A kit comprising: a plurality of primary antibodies, said plurality of primary antibodiescomprising a primary antibody specific to Ki67, a primary antibody specific to MUM1, a primary antibody specific to Bcl-2, a primary antibody specific to CD3, a primary antibody specific toLBR 20276-P-US CD10, a primary antibody specific to Cyclin D1 a primary antibody specific to CD20, a primary antibody specific to CD5, and a primary antibody specific to Bcl-6.

[0218] Example 47

[0219] The kit of example 46, further comprising a plurality of secondary antibodies, saidsecondary antibodies being conjugated to an enzyme selected from AP and HRP and specific to said plurality of antibodies.

[0220] Example 48

[0221] The kit of example 47, said plurality of secondary antibodies being anti-species secondaryantibodies.

[0222] Example 49

[0223] The kit of any of examples 46-48, further comprising a plurality of anti-species linkerantibodies specific to said plurality of primary antibodies.

[0224] Example 50

[0225] The kit of any of examples 46-49, further comprising a plurality of chromogens, saidplurality of chromogen comprising a brown chromogen, a red chromogen, and a green chromogen.

[0226] Example 51

[0227] The kit of any of examples 1-50, further comprising a buffered solution.V. Conclusion

[0228] It should be appreciated that any patent, publication, or other disclosure material, in wholeor in part, that is said to be incorporated by reference herein is incorporated herein only to the extent that the incorporated material does not conflict with existing definitions, statements, or other disclosure material set forth in this disclosure. As such, and to the extent necessary, the disclosure as explicitly set forth herein supersedes any conflicting material incorporated herein by reference. Any material, or portion thereof, that is said to be incorporated by reference herein, but whichLBR 20276-P-US conflicts with existing definitions, statements, or other disclosure material set forth herein will only be incorporated to the extent that no conflict arises between that incorporated material and the existing disclosure material.

[0229] Having shown and described various embodiments of the present invention, furtheradaptations of the methods and systems described herein may be accomplished by appropriate modifications by one of ordinary skill in the art without departing from the scope of the present invention. Several of such potential modifications have been mentioned, and others will be apparent to those skilled in the art. For instance, the examples, embodiments, geometrics, materials, dimensions, ratios, steps, and the like discussed above are illustrative and are not required. Accordingly, the scope of the present invention should be considered in terms of the following examples and is understood not to be limited to the details of structure and operation shown and described in the specification and drawings.

Claims

LBR 20276-P-US CLAIMS I / we claim:

1. A tissue staining method for characterization of non-Hodgkin’s lymphoma comprising:(a) performing, on a first tissue section, a plurality of immunohistochemistry (IHC) assayscomprising (i) a first IHC assay for detection of Ki67, said first IHC assay comprising a firstchromogen; (ii) a second IHC assay for detection of CD3, said second IHC assay comprising asecond chromogen; (iii) a third IHC assay for detection of CD20, said third IHC assay comprising a thirdchromogen; (b) performing, on a second tissue section, a plurality of immunohistochemistry assayscomprising (i) a first IHC assay for detection of MUM1, said first IHC assay comprising a firstchromogen; (ii) a second IHC assay for detection of CD10, said second IHC assay comprising asecond chromogen; (iii) a third IHC assay for detection of CD5, said third IHC assay comprising a thirdchromogen; (c) performing, on a third tissue section, an immunohistochemistry assay, saidimmunohistochemistry assay comprising (i) a first IHC assay for detection of Bcl-2, said first IHC assay comprising a firstchromogen; (ii) a second IHC assay for detection of CyclinD1, said second IHC assaycomprising a second chromogen; and (iii) a third IHC assay for detection of Bcl-6, said third IHC assay comprising a thirdchromogen.LBR 20276-P-US2. The method of any preceding claim, each said first assay being performed on said first tissuesection prior to said second assay, and each said second assay being performed prior to each said third assay.

3. The method of any preceding claim, said first IHC assay for detection of Ki67 comprisingcontacting said first tissue section with a primary antibody having specific binding to Ki67.

4. The method of any preceding claim, said second IHC assay for detection of CD3 comprisingcontacting said first tissue section with a primary antibody having specific binding to CD3.

5. The method of any preceding claim, said third IHC assay for detection of CD20 comprisingcontacting said first tissue section with a primary antibody having specific binding to CD20.

6. The method of any preceding claim, said first IHC assay for detection of MUM1 comprisingcontacting said first tissue section with a primary antibody having specific binding to MUM1.

7. The method of any preceding claim, said second IHC assay for detection of CD10 comprisingcontacting said first tissue section with a primary antibody having specific binding to CD10.

8. The method of any preceding claim, said third IHC assay for detection of CD5 comprisingcontacting said first tissue section with a primary antibody having specific binding to CD5.

9. The method of any preceding claim, said first IHC assay for detection of BCL2 comprisingcontacting said first tissue section with a primary antibody having specific binding to Bcl-2.

10. The method of any preceding claim, said second IHC assay for detection of CyclinD1comprising contacting said first tissue section with a primary antibody having specific binding to CyclinD1.

11. The method of any preceding claim, said third IHC assay for detection of Bcl-6 comprisingcontacting said first tissue section with a primary antibody having specific binding to Bcl-6.

12. The method of any of claims 3-11, said primary antibody being a monoclonal antibody.

13. The method of any of claims 3-12, said primary antibody being a mouse or rabbit monoclonalantibody.LBR 20276-P-US14. The method of any of claims 3-13, further comprising an anti-species linker, said anti-specieslinker being an antibody which specifically binds to said primary antibody.

15. The method of any of claims 1-14, further comprising a secondary antibody.

16. The method of claim 15, said secondary antibody being an anti-species monoclonal antibody.

17. The method of any of claims 14-15, said secondary antibody being an anti-species monoclonalantibody specific to said primary antibody.

18. The method of any of claims 14-16, said secondary antibody being an anti-species monoclonalantibody specific to said anti-species linker.

19. The method of any of claims 16-18, said anti-species monoclonal antibody comprising anenzyme for precipitating a chromogen.

20. The method of claim 19, said enzyme for precipitating a chromogen being selected fromalkaline phosphatase (AP) and / or horseradish peroxidase (HRP).

21. The method of any of claims 19-20, said first chromogen, said second chromogen, and saidthird chromogen reacting with said enzyme to form a precipitated product.

22. The method of claim 21, said precipitated product binding to tissues adjacent to where theprimary antibody is bound.

23. The method of any of claims 1-22, said first chromogen being a brown chromogen.

24. The method of any of claims 1-23, said second chromogen being a red chromogen.

25. The method of any of claims 1-24, said third chromogen being a green chromogen.

26. The method of claim 1 wherein said first, second, and third IHC assay comprise(a) contacting said tissue section with a primary antibody;LBR 20276-P-US (b) contacting said tissue sample with a secondary antibody conjugated to an enzymeconfigured to catalyze said chromogen, said secondary antibody being specific to said primary antibody; and (c) detecting said chromogen.

27. The method of claim 1 or 2, each said tissue section comprising tissue obtained from anindividual having, or suspected of having, non-Hodgkin’s lymphoma.

28. The method of any preceding claim, each said tissue section comprising tissue obtained fromthe same individual.

29. The method of any preceding claim, each said tissue section comprising tissue from the samebiopsy sample.

30. The method of any of claims 1-29, each said tissue section comprising tissue from differentbiopsy sample.

31. The method of any of claims 1-30, further comprising staining said first, second, and / or thirdtissue section with hematoxylin.

32. The method of any of claims 1-31, further comprising imaging said first, second, and / or thirdtissue section.

33. The method of any preceding claim, further comprising detecting said first chromogen, saidsecond chromogen, and said third chromogen.

34. The method of any of claims 1-33, wherein said first chromogen, said second chromogen, andsaid third chromogen are simultaneously detected.

35. An automated slide staining apparatus for assaying one or more tissue samples disposed on aslide, the apparatus comprising: (a) a controller;LBR 20276-P-US (b) a slide staining assembly, the slide staining assembly being configured to receive one or more of the slides; and (c) at least one fluid dispensing robot configured by the controller to dispense a plurality of reagents to the one or more slides received in the slide staining assembly to treat the one or more tissue samples respectively, the at least one fluid dispensing robot being configured by the controller to dispense one or more reagents of the plurality of reagents in a predetermined sequence corresponding to an IHC protocol for the one or more slides received in the slide staining assembly to treat the one or more tissue samples disposed on each slide independently, the at least one fluid dispensing robot being further configured by the controller to dispense a reagent for specific binding of Ki67, MUM1, or Bcl-2, and a first chromogen, a reagent for specific binding of CD3, CD10, or Cyclin D1 and a second chromogen, and a reagent for specific binding of CD20, CD5, and Bcl-6 and a third chromogen; in a predetermined sequence for performing a plurality of IHC staining protocols for the one or more slides received in the slide staining assembly to treat the one or more tissue samples disposed on each slide independently, and the at least one fluid dispensing robot being further configured by the controller to perform the predetermined sequence corresponding to the IHC staining protocol.

36. The apparatus claim 35, the slide staining assembly being configured by the controller to heateach slide received by the slide staining assembly independently to an incubation temperature.

37. The apparatus of claim 35 or 36, the slide staining assembly being configured by the controllerto heat each slide received by the slide staining assembly independently to an incubation temperature during the staining protocol.

38. A method for an automated multiplex IHC assay of one or more tissue samples disposed on aslide, the method comprising dispensing one or more reagents to the one or more tissue samples using one or more automated fluid dispensers in a series of staining steps associated with an IHCLBR 20276-P-US protocol, said reagents comprising a reagent for specific binding of Ki67, MUM1, or Bcl-2, and a first chromogen, a reagent for specific binding of CD3, CD10, or Cyclin D1 and a second chromogen, and a reagent for specific binding of CD20, CD5, and Bcl-6 and a third chromogen;.

39. The method of claim 38, further comprising dispensing one or more reagents to the one ormore tissue sections using one or more automated fluid dispensers in a series of sample preparation steps associated with a sample preparation protocol.

40. The method of any of claims 38-39, the one or more reagents comprising a primary antibodyspecific to Ki67, a primary antibody specific to MUM1, a primary antibody specific to Bcl-2, a primary antibody specific to CD3, a primary antibody specific to CD10, a primary antibody specific to Cyclin D1 a primary antibody specific to CD20, a primary antibody specific to CD5, and a primary antibody specific to Bcl-6.

41. The method of claim 40, the primary antibody comprising a mouse monoclonal antibody.

42. The method of any of claims 40-41, the primary antibody comprising a rabbit monoclonalantibody.

43. The method of any of claims 39-42, the one or more reagents comprising a secondary antibodyspecific to a primary antibody of claim 40.

44. The method of claim 43, the secondary antibody comprising an anti-species antibody.

45. The method of any of claims 43-44, the secondary antibody comprising an anti-mouse and / oranti-rabbit antibody.

46. A kit comprising:(a) a plurality of primary antibodies, said plurality of primary antibodies comprising aprimary antibody specific to Ki67, a primary antibody specific to MUM1, a primary antibody specific to Bcl-2, a primary antibody specific to CD3, a primary antibody specific to CD10, a primary antibody specific to Cyclin D1 a primary antibody specific to CD20, a primary antibody specific to CD5, and a primary antibody specific to Bcl-6.LBR 20276-P-US47. The kit of claim 46, further comprising a plurality of secondary antibodies, said secondaryantibodies being conjugated to an enzyme selected from AP and HRP and specific to said plurality of antibodies.

48. The kit of claim 47, said plurality of secondary antibodies being anti-species secondaryantibodies.

49. The kit of any of claims 46-48, further comprising a plurality of anti-species linker antibodiesspecific to said plurality of primary antibodies.

50. The kit of any of claims 46-49, further comprising a plurality of chromogens, said pluralityof chromogen comprising a brown chromogen, a red chromogen, and a green chromogen.

51. The kit of any of claims 1-50, further comprising a buffered solution.

Images