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WO2009085575A2 - Dispositif cellulaire étalonneur de contrôle qualité destiné à une épreuve biologique d'immunohistochimie et procédés d'utilisation de ce dispositif - Google Patents

Dispositif cellulaire étalonneur de contrôle qualité destiné à une épreuve biologique d'immunohistochimie et procédés d'utilisation de ce dispositif Download PDF

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Publication number
WO2009085575A2
WO2009085575A2 PCT/US2008/085841 US2008085841W WO2009085575A2 WO 2009085575 A2 WO2009085575 A2 WO 2009085575A2 US 2008085841 W US2008085841 W US 2008085841W WO 2009085575 A2 WO2009085575 A2 WO 2009085575A2
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cell pellet
positive control
control cell
antigen
sample
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PCT/US2008/085841
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WO2009085575A3 (fr
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Marc E. Key
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Spring Bioscience Corporation
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Publication of WO2009085575A3 publication Critical patent/WO2009085575A3/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/5436—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand physically entrapped within the solid phase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G01N1/31—Apparatus therefor
    • G01N1/312—Apparatus therefor for samples mounted on planar substrates
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/34—Microscope slides, e.g. mounting specimens on microscope slides

Definitions

  • This disclosure relates to immunohistochemistry, and specifically to methods of using a quality control device for an immunohistochemistry assay.
  • Immunohistochemistry is a technique involving the use of specific binding agents, such as antibodies, to detect specific antigens that may be present in a tissue sample.
  • IHC is widely used in clinical and diagnostic applications, such as to diagnose particular disease states or conditions. For example, a diagnosis of a particular type of cancer can be made based on the presence of a particular marker molecule present in a sample obtained from a subject.
  • IHC is also widely used in basic research to understand the distribution and localization of biomarkers in different parts of a tissue. A deficiency of IHC is its lack of standards, making accurate quantification of the results difficult, if not impossible.
  • the staining results achieved by IHC typically are scored subjectively, for instance on a four-point scale with 0 being negative and 4 being intensely positive. Because there are no standards for comparison, IHC is not quantitative, and there are no methods to correct for intra-run variability (the difference in staining between samples stained at the same time under similar conditions) and inter-run variability (the variability between slides that were stained at different times and possibly under different conditions). Thus, a number of potential errors can be introduced into the staining procedure, leading to erroneous results.
  • One embodiment of the method includes (i) providing a first universal positive control cell pellet section and a first test sample on same solid support, wherein the first universal positive control cell pellet section contains a known amount of a known antigen; (ii) performing immunohistochemistry on the first universal positive control cell pellet section and the first test sample; (iii) analyzing immunohistochemical staining intensity of the first universal positive control cell pellet section and the first test sample; (iv) assigning a numerical value to the immunohistochemical staining intensity of the first universal positive control cell pellet section; (v) assigning a numerical value to the immunohistochemical staining intensity of the first test sample; (vi) comparing the immunohistochemical staining intensity of the first universal positive control cell pellet section to an expected value for the first universal positive control cell pellet section, thereby providing a deviation between the immunohistochemical staining of the first universal positive control cell pellet section and the expected value; (vii) calculating a
  • Kits for performing IHC are also provided.
  • the kit can include one or more of the disclosed quality control devices.
  • the kit includes a slide that includes a universal cell pellet control section, instructions for carrying out the method, and/or at least one of a primary antibody and a secondary antibody.
  • FIG. 1 is a digital image of a quality control sample treated with a mouse monoclonal antibody to vimentin antigen illustrating a positive IHC reaction.
  • FIG. 2 illustrates a microscopic (1Ox) view of a test sample after IHC staining with vimentin antigen.
  • FIG. 3 shows a microscopic (4Ox) view of a universal positive control cell sample treated with a mouse monoclonal antibody to vimentin antigen in which the brown colored reaction illustrates positive staining.
  • FIG. 4 is a schematic diagram of a test tissue, a universal positive control cell pellet section, an antigen-retrieval control cell pellet section, and an endogenous peroxidase control cell pellet section placed on a solid support.
  • FIG. 5 provides a series of digital images and histograms illustrating staining variability in calibrator cells.
  • the upper left panel illustrates under-staining of calibrator cells, the upper center panel optimal staining and the upper right panel over-staining. Histograms beneath each panel illustrate the calculated staining for the calibrator cells.
  • FIG. 6 provides a series of digital images and histograms illustrating staining variability in test specimens.
  • the upper left panel illustrates under-staining, the upper center panel optimal staining and the upper right panel over-staining. Histograms beneath each panel illustrate the calculated staining for the calibrator cells.
  • FIG. 7 provides a series of digital images prior to (upper row) and following (lower row) removal of variability.
  • FIG. 8 provides a series of histograms representing calculated staining intensity of three test specimens after correction.
  • IHC immunohistochemistry scFv: single chain Fv proteins dsFv: disulfide stabilized Fv proteins VH: variable heavy
  • VL variable light
  • Antibody A polypeptide that includes at least a light chain or heavy chain immunoglobulin variable region and specifically binds an epitope of an antigen.
  • Antibodies include monoclonal antibodies, polyclonal antibodies, or fragments of antibodies as well as others known in the art.
  • an antibody is labeled with a detectable label such as an enzyme of fluorophore.
  • the term "specifically binds" refers to, with respect to an antigen, the preferential association of an antibody or other ligand, in whole or part, with a specific polypeptide.
  • a specific binding agent binds substantially only to a defined target. It is recognized that a minor degree of non-specific interaction may occur between a molecule, such as a specific binding agent, and a non-target polypeptide. Nevertheless, specific binding can be distinguished as mediated through specific recognition of the antigen. Although selectively reactive antibodies bind antigen, they can do so with low affinity.
  • Specific binding typically results in greater than 2- fold, such as greater than 5 -fold, greater than 10-fold, or greater than 100-fold increase in amount of bound antibody or other ligand (per unit time) to a target polypeptide, such as compared to a non-target polypeptide.
  • a variety of immunoassay formats are appropriate for selecting antibodies specifically immunoreactive with a particular protein.
  • solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York (1988), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.
  • Antibodies are composed of a heavy and a light chain, each of which has a variable region, termed the variable heavy (VH) region and the variable light (VL) region. Together, the VH region and the VL region are responsible for binding the antigen recognized by the antibody.
  • VH region and VL region are responsible for binding the antigen recognized by the antibody.
  • a scFv protein is a fusion protein in which a light chain variable region of an immunoglobulin and a heavy chain variable region of an immunoglobulin are bound by a linker, while in dsFvs, the chains have been mutated to introduce a disulfide bond to stabilize the association of the chains.
  • the term also includes recombinant forms such as chimeric antibodies (for example, humanized murine antibodies), heteroconjugate antibodies (such as, bispecific antibodies). See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, Immunology, 3rd Ed., W.H. Freeman & Co., New York, 1997.
  • a “monoclonal antibody” is an antibody produced by a single clone of B-lymphocytes or by a cell into which the light and heavy chain genes of a single antibody have been transfected.
  • Monoclonal antibodies are produced by methods known to those of skill in the art, for instance by making hybrid antibody- forming cells from a fusion of myeloma cells with immune spleen cells. These fused cells and their progeny are termed "hybridomas.”
  • Monoclonal antibodies include humanized monoclonal antibodies.
  • Antigen A molecule that stimulates an immune response. Antigens are usually proteins or polysaccharides. An epitope is an antigenic determinant. These are particular chemical groups or peptide sequences on a molecule that are antigenic, such that they elicit a specific immune response. An antibody binds a particular antigenic epitope. The binding of an antibody to a particular antigen or epitope of an antigen can be used to localize the position of the antigen for example in or on a bio logical sample, or determine if the particular antigen is present in a biological sample. An antigen of interest is an antigen for which the IHC assay is designed to detect in a test sample.
  • Antigen retrieval A process for recovering antigenicity in fixed processed tissue samples. Antigen retrieval is also sometimes referred to as epitope retrieval, target retrieval, or target unmasking. Various methods are used for antigen retrieval, including heat treatment, protease digestion, or a combination of heat and protease treatment.
  • Antigen-retrieval control Refers to a cell pellet that serves as a positive control to confirm that an antigen-retrieval procedure was performed as part of the immunohistochemistry assay. In one example, the antigen-retrieval control cells express mouse immunoglobulin.
  • Antigen-specific control refers to a cell pellet in which the cells are known to express an antigen of interest such that when an IHC assay is performed, the antigen-specific control produces a positive signal.
  • Binding or stable binding An association between two substances or molecules, such as the association of a specific binding agent (e.g., antibody) with an antigen.
  • a specific binding agent e.g., antibody
  • Binding affinity The tendency of one molecule to bind (typically non- covalently) with another molecule, such as the tendency of one member of a specific binding pair to bind with another member of a specific binding pair.
  • a binding affinity can be measured as a binding constant, which binding affinity for a specific binding pair (such as an antibody/antigen pair or nucleic acid probe/nucleic acid sequence pair) can be at least 1 x 10 5 M "1 , such as at least 1 x 10 6 M "1 , at least 1 x 10 7 M “1 or at least 1 x 10 8 M "1 .
  • Binding affinity can calculated by a modification of the Scatchard method described by Frankel et al. (MoI.
  • a high binding affinity can be measured by a competition radioimmunoassay.
  • a high binding affinity for an antibody/antigen pair is at least about 1 x 10 8 M "1 .
  • a high binding affinity is at least about 1.5 x 10 8 M “1 , at least about 2.0 x 10 8 M “1 , at least about 2.5 x 10 8 M “1 , at least about 3.O x IO 8 M “1 , at least about 3.5 x 10 8 M “1 , at least about 4.0 x 10 8 M “1 , at least about 4.5 x 10 8 M “1 , or at least about 5.0 x 10 8 M “1 .
  • Carrier A molecule to which a hapten or an antigen can be bound.
  • Carrier molecules include immunogenic carriers and specific-binding carriers. When bound to an immunogenic carrier, the bound molecule may become immunogenic.
  • Immunogenic carriers may be chosen to increase the immunogenicity of the bound molecule and/or to elicit antibodies against the carrier, which are diagnostically, analytically, and/or therapeutically beneficial. Covalent linking of a molecule to a carrier can confer enhanced immunogenicity and T-cell dependence (Pozsgay et al , PNAS 96:5194-97, 1999; Lee et al, J. Immunol 116: 1711-18, 1976; Dintzis et al, PNAS 73:3671-75, 1976).
  • Useful carriers include polymeric carriers, which can be natural (for example, proteins from bacteria or viruses), semi-synthetic or synthetic materials containing one or more functional groups to which a reactant moiety can be attached. Specific binding carriers can by any type of specific binding moiety, including an antibody, a nucleic acid, an avidin, a protein-nucleic acid.
  • suitable immunogenic carriers are those that can increase the immunogenicity of a hapten and/or help elicit antibodies against the hapten which are diagnostically, analytically, and/or therapeutically beneficial.
  • Useful carriers include polymeric carriers, which can be natural (such as proteins like ovalbumin or keyhole limpet hemocyanin) or derived from a natural polymer isolated from any organism (including viruses), semi-synthetic or synthetic materials containing one or more functional groups, for example primary and/or secondary amino groups, azido groups, hydroxyl groups, or carboxyl groups, to which a reactant moiety can be attached.
  • the carrier can be water soluble or insoluble, and can be a protein or polypeptide.
  • Carriers that fulfill these criteria are generally known in the art (see, for example, Fattom et al, Infect. Immun. 58:2309-12, 1990; Devi et al, PNAS 88:7175-79, 1991; Szu et al, Infect. Immun. 59:4555-61, 1991; Szu et al, J. Exp. Med. 166: 1510-24, 1987; and Pavliakova et al, Infect. Immun. 68:2161-66, 2000).
  • Cell pellet A collection of cells that has been compressed in volume to form a densely packed mass of cells. Cell pellets are typically formed by centrifugation of a collection of cells, such as cells suspended in growth medium.
  • Conjugating, joining, bonding or linking Covalently linking one molecule to another molecule, or to a cell or tissue.
  • conjugating, joining, bonding or linking includes making two polypeptides into one contiguous polypeptide molecule, or covalently attaching a hapten or other molecule to the surface of a cell.
  • the linkage is by chemical means.
  • “Chemical means” refers to a reaction between the hapten and the cell such that there is a covalent bond formed.
  • Placement in direct physical association for example both in solid form and/or in liquid form (for example, the placement of a biological sample, such as a biological sample affixed to a slide, in contact with an antigen releasing solution).
  • Control A sample or procedure performed to assess test validity.
  • a control is a quality control, such as a positive control.
  • a positive control is a procedure or sample, such as a tissue or cell, that is similar to the actual test sample, but which is known from previous experience to give a positive result. The positive control confirms that the basic conditions of the test produce a positive result, even if none of the actual test samples produce such result.
  • a positive control is a sample known by previous testing to contain the suspected antigen.
  • a control is a negative control.
  • a negative control is a procedure or test sample known from previous experience to give a negative result. The negative control demonstrates the base-line result obtained when a test does not produce a measurable positive result; often the value of the negative control is treated as a "background" value to be subtracted from the test sample results.
  • a negative control is a reagent that does not include the specific primary antibody.
  • calibrator controls which are universal positive controls that contain a known amount of the universal positive control antigen. Such calibrator controls have an expected signal intensity, and therefore can be used to correct for inter- or intra-run staining variability.
  • an antigen "coupled” to a cell means the antigen is attached to the cell by chemical, physical or passive means.
  • the antigen is coupled to the cell by chemical conjugation. Methods of conjugation are well known in the art (see, for example, Hermanson, G. T., “Bioconjugate Techniques,” Academic Press, San Diego, CA, 1996).
  • Covalently linked refers to a covalent linkage between atoms by the formation of a covalent bond characterized by the sharing of pairs of electrons between atoms.
  • a detectable label is covalently linked to a specific binding agent, such as an antibody.
  • Detect To determine if an agent (such as a signal or particular antigen or protein) is present or absent, for example, in a sample. In some examples, this can further include quantification.
  • Detectable Label A detectable compound or composition that is attached directly or indirectly to another molecule, such as an antibody or a protein, to facilitate detection of that molecule. Specific, non- limiting examples of labels include fluorophores, enzymes, and radioactive isotopes.
  • Dispenser A unit capable of dispensing or providing something.
  • a dispenser is an object that supports or contains a device, such as a quality control device, and has a mechanism to detach or dispense the device.
  • a dispenser is configured to store multiple quality control devices and allow individual units of such devices to be efficiently dispensed, for example, by including a cutting mechanism (e.g., a plurality of teeth) at one end of the device.
  • Endogenous antigen An antigen synthesized within a cell. In one embodiment, the endogenous antigen is an immunoglobulin molecule.
  • Endogenous peroxidase control refers to a sample, such as a cell pellet, that serves as a control for endogenous peroxidase activity. If an IHC assay uses peroxidase as a means of assessing the presence of the antigen of interest, the test and control samples are blocked to eliminate endogenous peroxidase activity of the cells or tissue. A positive result indicates the sample was not properly blocked against endogenous peroxidase activity. A negative result indicates peroxidase activity was effectively blocked, thus the IHC assay is valid.
  • Endogenous peroxidase control cells include cells having peroxidase activity, or pseudo- peroxidase activity. Such cells include, but are not limited to, cells of hematogenous origin, such as red blood cells.
  • Epitope An antigenic determinant. These are particular chemical groups or contiguous or non-contiguous peptide sequences on a molecule that are antigenic, that is, that elicit a specific immune response. An antibody binds a particular antigenic epitope.
  • Exogenous antigen An antigen coupled to the exterior of a cell. In one embodiment, the exogenous antigen is a hapten, such as fluorescein, biotin, dinitro phenol or digoxigenin.
  • Fixation A process which preserves cells and tissue constituents in as close to a life-like state as possible and allows them to undergo preparative procedures without change. Fixation arrests the autolysis and bacterial decomposition processes which begin upon cell death, and stabilizes the cellular and tissue constituents so that they withstand the subsequent stages of tissue processing, such as for IHC.
  • Tissues may be fixed by either perfusion with or submersion in a fixative, such as an aldehyde (such as formaldehyde, paraformaldehyde, glutaraldehyde, and the like).
  • fixatives include oxidizing agents (for example, metallic ions and complexes, such as osmium tetroxide and chromic acid), protein-denaturing agents (for example, acetic acid, methanol, and ethanol), fixatives of unknown mechanism (for example, mercuric chloride, acetone, and picric acid), combination reagents (for example, Carnoy's fixative, methacarn, Bouin's fluid, B5 fixative, Rossman's fluid, and Gendre's fluid), microwaves, and miscellaneous (for example, excluded volume fixation and vapour fixation).
  • Additives may also be included in the fixative, such as buffers, detergents, tannic acid, phenol, metal salts (for
  • formaldehyde generally in the form of a formalin solution (4% formaldehyde in a buffer solution, referred to as 10% buffered formalin).
  • Fluorophore A chemical compound, which when excited by exposure to a particular stimulus, such as a defined wavelength of light, emits light (fluoresces), for example at a different wavelength (such as a longer wavelength of light). Fluorophores are part of the larger class of luminescent compounds. Luminescent compounds include chemiluminescent molecules, which do not require a particular wavelength of light to luminesce, but rather use a chemical source of energy. Therefore, the use of chemiluminescent molecules (such as aequorin) can eliminate the need for an external source of electromagnetic radiation, such as a laser. Examples of particular fluorophores that can be used in the methods disclosed herein are provided in U.S. Patent No.
  • fluorophores include those known to those skilled in the art, for example those available from Molecular Probes (Eugene, OR).
  • a fluorophore is used as a donor fluorophore or as an acceptor fluorophore.
  • Hapten A molecule, typically a small molecule that can combine specifically with an antibody, but typically is substantially incapable of being immunogenic except in combination with a carrier molecule. Examples of haptens include, but are not limited to fluorescein, biotin, dinitrophenol, and digoxigenin.
  • High throughput technique Methods that allow rapid IHC staining of multiple samples, for example, through a combination of robotics, data processing and control software, liquid handling devices, and sensitive detectors, high throughput techniques allows the rapid IHC staining of multiple samples.
  • Immunohistochemistry A method of determining the presence or distribution of an antigen in a sample by detecting interaction of the antigen with a specific binding agent, such as an antibody.
  • Label An agent capable of detection, for example by ELISA, spectrophotometry, flow cytometry, or microscopy.
  • a label can be attached to a specific binding agent, such as an antibody, thereby permitting detection of the specific binding agent and hence an antigen bound by the specific binding agent.
  • labels include, but are not limited to, radioactive isotopes, enzyme substrates, co-factors, ligands, chemiluminescent agents, fluorophores (such as small molecule fluorophores or semiconductor nanocrystals), haptens, enzymes, and combinations thereof.
  • Membrane A support surface on which a sample can be immobilized.
  • a membrane is any surface that allows a sample, such as a quality control sample, to be immobilized and subsequently removed without significantly damaging the cellular components ⁇ e.g., causing the sample to wrinkle, tear and the like) of the sample.
  • a membrane includes a first surface that is a detachable surface.
  • a detachable surface is any surface from which a sample can be detached from without significantly damaging the cellular components. Examples of a detachable surface include regenerated cellulose ⁇ e.g., cellophane), paraffin, wax, and fluoropolymer coatings, such as polytetrafluoroethylene and perfluoroalkoxy.
  • the membrane is a membrane "strip" which includes multiple quality control samples, such as multiple control cell pellet sections.
  • the membrane strip includes perforations assisting with the separation of individual quality control devices.
  • Polymeric substance A substance composed of molecules with large molecular mass composed of repeating structural units, or monomers, connected by covalent chemical bonds. As used herein, examples of polymeric substances can include paraffin, agarose, and gelatin.
  • Quality control device A device employed to assess assay validity, such as
  • a quality control device is a device useful for monitoring quality control of assays that measure analytes in cells or tissue samples obtained from biological samples.
  • a quality control device includes a membrane and a quality control sample, such as a universal positive control cell pellet section.
  • a sample such as a biological sample, includes biological materials (such as nucleic acid and proteins, for example double-stranded nucleic acid binding proteins) obtained from an organism or a part thereof, such as a plant, animal, bacteria, and the like.
  • the biological sample is obtained from an animal subject, such as a human subject.
  • a biological sample is any solid or fluid sample obtained from, excreted by or secreted by any living organism, including without limitation, single celled organisms, such as bacteria, yeast, protozoans, and amebas among others, multicellular organisms (such as plants or animals, including samples from a healthy or apparently healthy human subject or a human patient affected by a condition or disease to be diagnosed or investigated, such as cancer).
  • a biological sample can be a biological fluid obtained from, for example, blood, plasma, serum, urine, bile, ascites, saliva, cerebrospinal fluid, aqueous or vitreous humor, or any bodily secretion, a transudate, an exudate (for example, fluid obtained from an abscess or any other site of infection or inflammation), or fluid obtained from a joint (for example, a normal joint or a joint affected by disease).
  • a biological sample can also be a sample obtained from any organ or tissue (including a biopsy or autopsy specimen, such as a tumor biopsy) or can include a cell (whether a primary cell or cultured cell) or medium conditioned by any cell, tissue or organ.
  • a biological sample is a nuclear extract.
  • a biological sample is bacterial cytoplasm.
  • a sample is a quality control sample, such as one of the disclosed cell pellet section samples.
  • a sample is a test sample.
  • a test sample is a cell, a tissue or cell pellet section prepared from a biological sample obtained from a subject.
  • the subject is one that is at risk or has acquired a particular condition or disease.
  • Solid support Any solid surface suitable for processing IHC samples, such as one or more of the disclosed control cell pellet samples and a test sample.
  • a solid support surface is a rigid, flat surface.
  • a solid support can be a rigid, flat, optically opaque and substantially non-fluorescent surface, such as a glass microscope slide.
  • the solid support fits entirely onto the stage of a microscope and accommodates at least one quality control sample and test sample to allow simultaneous processing.
  • the solid support is any surface to which a sample can be permanently attached or affixed.
  • a solid support can include a non-detachable surface (e.g., a surface to which a sample cannot be removed from without causing significant damage to the sample).
  • Tissue A collection of interconnected cells that perform a similar function within an organism.
  • Universal positive control A cell (or a cell pellet) that expresses a known endogenous antigen or includes a known exogenous antigen coupled to the cell surface.
  • the known antigen of the universal positive control is structurally unrelated to the antigen of interest, but always stains positive when the IHC procedure is performed correctly.
  • the known antigen is an immunoglobulin.
  • a positive result with the universal positive control indicates that secondary antibody (e.g., anti- immunoglobulin antibody) treatment and all subsequent steps of the IHC assay were performed correctly.
  • the known antigen is an exogenous antigen, such as fluorescein.
  • a positive result with the universal positive control indicates that primary antibody (e.g., anti-fluorescein antibody) treatment and all subsequent steps of the IHC assay were performed correctly.
  • primary antibody e.g., anti-fluorescein antibody
  • II. Introduction When using IHC, for example to analyze suspected disease tissue, typically the following procedures are performed: (1) isolate the desired tissue; (2) process the tissue in preparation for IHC analysis (which includes putting the tissue on a first microscope slide); (3) obtain another microscope slide containing a sample that can serve as a positive control, typically an antigen specific positive control; (4) stain both slides by IHC; and (5) examine both slides (e.g., using a microscope). If the antigen is detected in the positive control sample, then the test is considered to be valid.
  • the target antigen e.g., antigen known to be a marker for a suspected disease
  • the test tissue e.g., suspected disease tissue
  • the presence of the target antigen is confirmed (e.g., confirming presence of the disease); if the test tissue is negative, the presence of the target antigen is not confirmed (e.g., the disease is not confirmed).
  • a disadvantage of the positive control sample typically used in IHC is that the test sample is on one slide and the positive control sample is on another slide. Therefore, there are a number of potential errors that could be introduced into the staining procedure that would not be detected by the positive control sample. This often results in intra-run variability, such as one of the slides staining slightly stronger or slightly weaker than the other slide. Another source of error occurs when one test sample is compared to another test sample. Sometimes it is necessary to test multiple tissue samples simultaneously. In such instance, usually only a single positive control sample is run. Because of the known intra-run variability among samples, such comparisons are often not accurate.
  • a positive control sample on same microscope slide as the test sample.
  • a test sample which contains its own control on the same slide results in the test sample having its own internal control (e.g., when the slide is stained, both the test sample and the positive control sample are stained at the same time under identical conditions).
  • the reagents and the incubation times are identical. If the slide is over- stained or under-stained, the positive control sample will be similarly affected and any over- or under-staining will be immediately noticeable in the positive control sample.
  • the present disclosure solves one or more of the aforementioned problems by providing positive control cell pellet sections that are fixed or attached onto a detachable/releasable surface and not onto a glass microscope slide.
  • the control cell pellet samples of the present disclosure can be peeled off of the detachable support material and transferred onto non-detachable surface (such as a glass microscope slide) for analysis.
  • These control cell pellet sections are described as "Peel-and- Stick.”
  • control cell pellets and their methods of use.
  • universal positive control cell pellets, antigen-retrieval control cell pellets and endogenous peroxidase control cell pellets are described. Uses are also disclosed for these three control cell pellets in immunohistochemistry procedures.
  • calibrator cell pellets and methods of use as a standard for quantifying the intensity of immunohistochemical staining for instance for normalizing immunohistochemical staining to a calibrated standard, thereby allowing more accurate quantitation of staining intensity.
  • a calibrator cell pellet can contain a known quantity of a universal positive control antigen, and therefore produces an expected staining intensity. Thus, it is useful for correcting for both inter- and intra- run variability in IHC assays. Other embodiments are kits for performing the assay. III. Control Cell Pellets
  • control cell pellets described herein include the universal positive control cell pellet, the antigen-retrieval control cell pellet and the endogenous peroxidase control cell pellet. These three control cell pellets can be used individually or they can be used together, in any combination, as part of an array for simultaneous use in an IHC assay.
  • IHC assays typically include one or more controls to ensure that each step of the method was performed correctly and the reagents are functioning properly.
  • a standard assay includes a negative control and a positive control.
  • a negative control is usually a reagent that does not contain the specific primary antibody.
  • the negative control reagent is usually placed on a second microscope slide which contains a second portion of the test sample.
  • a positive control is usually a tissue that is known by previous testing to contain the antigen of interest. The positive control always produces a positive colored end-product. If the positive control stains negative, then the test is considered invalid.
  • Cell pellets are formed from cell lines and differ from tissues in several respects. Specifically, cell lines (i) are grown in vitro; (ii) are uniform with respect to their biological properties and antigen expression; (iii) provide a limitless supply of control materials; and (iv) are well characterized in terms of their characteristics, including quantitation of antigen levels. Because of these properties, control cell pellets offer advantages over other types of controls for IHC assays.
  • the universal positive control cell pellet contains a generic antigen that is not structurally related to the antigen of interest, but nevertheless always stains positive when the IHC procedure has been performed correctly. This provides the same guarantee that the test was valid as does an antigen-specific positive control cell pellet (a cell pellet including cells known to express the antigen of interest).
  • antigen-specific positive control cells One of the major limitations of the currently available antigen-specific positive control cells is that they cannot be used universally for all IHC procedures. Currently, only a small number of antigen-specific positive control cells are currently available. However, in IHC there are hundreds of different antigens that are routinely tested for, and for most of these antigens, an antigen-specific positive control cell pellet is not commercially available. Thus, provided herein are universal positive control cell pellets and uses thereof.
  • Antigen retrieval is a process involving heating a solid support, such as a microscope slide, containing a test sample to an elevated temperature ⁇ e.g. , about
  • the present disclosure provides an antigen-retrieval control cell pellet that can definitively confirm that antigen-retrieval was performed properly.
  • the antigen-positive cells are labeled with a detectable marker ⁇ e.g., that can be observed under the microscope).
  • a detectable marker ⁇ e.g., that can be observed under the microscope.
  • One common method of labeling cells is with the enzyme peroxidase. After the cells have been labeled with peroxidase they are next exposed to a colorless substrate solution, such as diaminobenzidine (DAB). If peroxidase is present, DAB is oxidized to a brown insoluble reaction product that acts to dye the cells. The dyed cells can be readily observed under the microscope. In contrast, if the cells are not labeled with peroxidase, the substrate is not oxidized, and the cells remain unstained.
  • DAB diaminobenzidine
  • the IHC process can include a step which blocks endogenous peroxidase. Before application of the primary antibody, endogenous peroxidase is blocked by incubation of the test sample in a peroxidase blocking solution, such as 3% hydrogen peroxide. This causes any endogenous peroxidase to be destroyed. At the end of the IHC staining procedure, any brown staining observed comes from the peroxidase-labeled cells, since there was no endogenous source of peroxidase.
  • the present disclosure provides an endogenous peroxidase control cell pellet that can definitively identify the status of a slide with respect to its endogenous peroxidase blocking.
  • the cell pellet stains positive when peroxidase blocking was inadequate and stains negative when peroxidase blocking was successfully applied.
  • IHC assays use the enzyme alkaline phosphatase, which is also present in mammalian cells as an endogenous enzyme.
  • IHC assays using alkaline phosphatase generally include a blocking step to eliminate this endogenous activity.
  • control cell pellets can also be made using cells that express endogenous alkaline phosphatase.
  • the method includes selecting a universal positive control cell line, wherein cells of the cell line express a known antigen; expanding the cells in vitro; and collecting and concentrating the cells into a pellet.
  • the conditions and procedures used for expanding cells in vitro will vary depending on the cell line selected as the universal positive control; however, cell culture methods are well known in the art. Methods of collecting the cells and concentrating the cells into a pellet, such as by centrifugation, also are well known in the art.
  • the antigen is an endogenous antigen.
  • the endogenous antigen is mouse immunoglobulin.
  • Cell lines expressing mouse immunoglobulin include, but are not limited to, hybridoma cell lines, lymphoma cell lines and plasmacytoma cell lines. Numerous cell lines expressing mouse immunoglobulin are known and are publicly available, such as from the American Type Culture Collection (Manassas, VA).
  • the method includes labeling cells in vitro with an exogenous antigen and collecting and concentrating the cells into a pellet.
  • the exogenous antigen is coupled to the cells.
  • Means of coupling the antigen to the cells include, but are not limited to chemical conjugation and physical means. Conjugation techniques are well known in the art (see, for example, Hermanson, G. T., "Bioconjugate Techniques," Academic Press, San Diego, CA, 1996).
  • the exogenous antigen can be any antigen for which a specific antibody exists or can be generated.
  • the exogenous antigen is a protein.
  • the exogenous antigen is a hapten.
  • Haptens suitable for use in the methods disclosed herein include, but are not limited to fluorescein, biotin, dinitro phenol and digoxigenin.
  • the disclosed methods for preparing a universal positive control cell pellet can further include one or more of the following steps: fixing the cell pellet; embedding the cell pellet into a polymeric substance; cutting a section of the embedded cell pellet; and placing the section on a solid support. Methods for fixing and embedding cells, as well as cutting sections of the embedded cell pellets, are described herein and are well known in the art. As described herein, the cell pellets are embedded in a polymeric substance.
  • any polymeric substance can be used as long as it allows for preparing thin sections of the embedded cell pellets ⁇ e.g., the material provides enough rigidity to allow the sample to be sectioned).
  • the polymeric substance is paraffin.
  • the polymeric substance is agarose.
  • the polymeric substance is gelatin.
  • the cells can be suspended in a liquid media, such as hot agar, and dispensed onto a detachable surface by a dispensing device, such as a pipette.
  • a dispensing device such as a pipette.
  • the solid support can be any solid surface suitable for processing IHC samples.
  • the solid support is a glass slide.
  • the solid support further includes a test sample, an antigen retrieval control cell pellet section, an endogenous peroxidase control cell pellet, or a combination thereof.
  • control cell pellets are embedded in and cut from the same polymeric block before placing on the solid support.
  • the methods can include fixing a universal positive control cell pellet and test sample, wherein the test sample is a tissue or cell pellet; embedding the universal positive control cell pellet and test sample into a polymeric substance; cutting a section from the embedded universal positive control cell pellet and the embedded test sample; and placing the sections on the same solid support.
  • the method further includes preparing a universal positive control cell pellet, including selecting a universal positive control cell line, wherein the cells of the cell line express a known antigen; expanding the cells in vitro; and collecting and concentrating the cells into a pellet.
  • the method further includes preparing a universal positive control cell pellet, including labeling cells in vitro with an exogenous antigen; and collecting and concentrating the cells into a pellet.
  • the solid support further includes an antigen retrieval control cell pellet section, an endogenous peroxidase control cell pellet, or both.
  • the control cell pellets are embedded in the same polymeric block.
  • the antigen is an endogenous antigen.
  • the endogenous antigen is mouse immunoglobulin.
  • the antigen is an exogenous antigen.
  • the exogenous antigen can be coupled to the cells, such as by chemical or physical means.
  • the exogenous antigen is a hapten.
  • the devices provide a quality control sample, such as a positive control cell pellet section, on a membrane so that the quality control sample can be removed from such surface and transferred onto a second surface (e.g. , a glass microscope slide) containing a test sample.
  • a quality control sample such as a positive control cell pellet section
  • a second surface e.g. , a glass microscope slide
  • the disclosed device allows each test sample to contain its own internal positive control (e.g., a quality control sample positioned and processed simultaneously on the same slide as the test tissue).
  • the disclosed quality control device includes a quality control sample and a membrane.
  • the quality control sample includes control cells embedded in a polymeric substance.
  • any polymeric substance can be used as long as allows for preparing thin sections (e.g., about 2 to 8 microns) of the embedded samples.
  • the polymeric substance is paraffin.
  • the polymeric substance is agarose.
  • the polymeric substance is gelatin.
  • the quality control sample is a tissue or cell pellet section, such as one of the disclosed control cell pellet sections.
  • the quality control sample is a universal positive control cell pellet sample.
  • a universal positive control cell pellet section can be formed of cells expressing an endogenous antigen, such as an immunoglobulin.
  • the quality control sample is a universal positive control cell pellet section formed of cells labeled with an exogenous antigen, such as fluorescein, biotin, digoxigenin, or dinitro phenol.
  • the exogenous antigen can be coupled to the cells such as by chemical or physical means.
  • the exogenous antigen is chemically conjugated to the cells.
  • the quality control sample is an antigen retrieval control cell pellet section.
  • the quality control sample is an endogenous peroxidase control cell pellet sample.
  • the size and the shape of the quality control sample can vary, for example the quality control sample can be circular, square, oval, rectangular or irregular in shape.
  • the sample is of a size that allows the quality control sample to be processed on a standard microscope slide of approximately 1 inch x 3 inches.
  • the quality control sample is smaller than 1 inch x 3 inches, such as 0.1 inches to 0.9 inches x 0.1 inches to 2 inches.
  • the sample size permits the control sample be placed adjacent a test sample on the same solid support (e.g., a microscope slide), such as size of approximately 1 to 2 millimeters in diameter.
  • the quality control sample is a microtome section of approximately 2 or more microns thick, such as at least 4, at least 6 or at least 8 microns.
  • the disclosed quality control device includes a membrane.
  • the membrane can be any surface that allows a sample, such as a quality control sample, to be immobilized and subsequently removed without significantly damaging the cellular components of the sample (e.g., causing the sample to wrinkle, tear and the like).
  • a membrane includes a first surface that is a detachable surface.
  • a detachable surface is any surface from which a sample can be detached from without significantly damaging the cellular components. Examples of a detachable surface include regenerated cellulose (e.g., cellophane), cellulose acetate, nylon, polycarbonate, paraffin, wax, and fluoropolymer coatings, such as polytetrafluoroethylene and perfluoroalkoxy.
  • the detachable surface is regenerated cellulose.
  • the detachable surface is paraffin.
  • the detachable surface is a surface coated with a fluropolymer.
  • the membrane is a membrane "strip" which includes multiple quality control samples, such as multiple control cell pellet sections.
  • the membrane strip includes perforations assisting with the detachment of individual quality control devices. It is however readily apparent that other shapes or forms of the membrane can be used with the quality control device, and these alternative forms are also encompassed by the present disclosure.
  • the quality control device includes a dispenser that is operationally coupled to the membrane for allowing the membrane with the quality control sample to be efficiently dispensed, for example by a user.
  • the size and shape of the dispenser can vary so long as the dispenser is capable of supporting a quality control device and has a mechanism to detach or dispense the device.
  • a dispenser is configured to store multiple quality control devices and allow individual units of such devices to be efficiently dispensed.
  • a dispenser can be designed so that individual quality control devices can be peeled from a membrane strip, such as a membrane strip that had been rolled, in a manner similar to removing a postage stamp from a long row (e.g., roll) of stamps.
  • a dispenser includes a cutting mechanism (e.g., a plurality of teeth) on one end of the device allowing individual quality control devices to be separated from the remaining roll of quality control devices.
  • a cutting mechanism e.g., a plurality of teeth
  • Additional examples of a dispenser can include a dispenser that supports multiple individual strips of membranes each including a quality control sample, so that individual quality control devices can be dispensed without use of a serrated edge.
  • the second surface to which the quality control sample is adhered is a non-detachable surface for permanently adhering the quality control sample.
  • the second surface can be a solid support surface (e.g., a surface sufficiently rigid to provide support to a sample).
  • the solid support can be any solid surface suitable for processing IHC samples.
  • a solid support is a rigid, flat, optically opaque and substantially non- fluorescent surface, such as a glass microscope slide or multi-well plates.
  • the solid support fits entirely onto the stage of a microscope and accommodates at least one quality control sample and test sample to allow simultaneous processing.
  • the solid support can include a test sample, a universal positive control cell pellet sample, an antigen retrieval control cell pellet sample, an endogenous peroxidase control cell pellet sample or combinations thereof.
  • a feature of this disclosure is to prepare a quality control sample, such as a cell pellet section, in a way that it can first be attached to a surface in a temporary manner, and later removed (e.g., peeled off) from this temporary surface and attached to another permanent surface.
  • a quality control sample such as a cell pellet section
  • the rationale for this approach has been more fully explained above.
  • a goal of the present disclosure is to provide a device that allows each test sample to contain its own internal positive control (e.g. , a quality control sample positioned and processed simultaneously on the same slide as the test sample).
  • methods for preparing a quality control device can include immobilizing a quality control sample on a first surface, in which the quality control sample includes a cell pellet section and the first surface is a detachable surface.
  • the methods also involve preparing the quality control sample by selecting a quality control sample cell line, wherein the cells of the cell line express a known antigen; expanding the cells in vitro; and collecting and concentrating the cells into a cell pellet.
  • the methods include removing the quality control sample from the first surface.
  • the methods can also include adhering the quality control sample to a second surface, such as a glass microscope slide. A detailed description for such methods is described elsewhere herein.
  • a calibrator cell pellet (also referred to as calibrator cells) that is useful for calibrating IHC staining intensity.
  • a deficiency of IHC is its lack of standards, making accurate quantitation difficult, if not impossible.
  • the staining results achieved by IHC are typically viewed by a trained observer under the microscope, and the staining intensity is subjectively scored usually on a scale of 0 to 4, with 0 being negative and 4 being intensely positive. Because there are no standards for comparison, prior to this disclosure, the method was not quantitative, and there were no methods to correct for intra- and inter-run variability. Because of this variability, a number of potential errors can be introduced into the staining procedure. If these errors are undetected, they may lead to erroneous results.
  • errors are technical errors that arise when two or more slides are unintentionally treated differently during the staining protocol.
  • the most common source of error is inconsistency in the incubation time following application of one or more IHC reagents to different slides. Even small inconsistencies in incubation time can result in one of the slides staining slightly more strongly or slightly more weakly than another slide.
  • This type of error is known as intra-run variability (e.g., the difference in staining between slides that were all stained at the same time under more or less similar conditions.)
  • Another source of error occurs when one test tissue is compared to another test tissue. Sometimes it is necessary for a laboratory to test multiple patient tissue samples simultaneously. However, because of the known intra-run variability among samples such comparisons are not always accurate.
  • Inter-run variability is the variability between slides that were stained at different times and possibly under different conditions. Inter-run variability is usually greater than intra-run variability.
  • Calibrator cells provide a fixed standard to which the IHC staining results are compared. IHC staining results are then normalized to the calibrated standards such that intra- and inter-run variability are significantly reduced, or even eliminated.
  • a calibrator cell pellet standard is similar to the positive control described above in that it contains the molecule of interest. However, a calibrator cell pellet standard also has a known amount of the molecule (e.g., antigen) of interest. Frequently, a quantitative assay will contain more than one standard with each standard having a different (but known) amount of the molecule of interest. When the staining is completed, each of the standards will exhibit a color which is proportional to the amount of the molecule contained in that standard. By first measuring the amount of color (such as in a spectrophotometer, for example) a graph can be made by plotting a standard curve of the optical density (color) of the standard on the Y-axis and the molecule concentration of the X-axis.
  • the amount of color such as in a spectrophotometer, for example
  • Calibrator cells provide essentially the same function for IHC that standards provide for quantitative diagnostic tests.
  • the calibrator cells provide an expected staining result, and thus have an expected value.
  • a calibrator cell pellet is placed on each microscope slide with the test tissue to be stained, and thus becomes physically linked to the test specimen.
  • the slide is then stained by the IHC method, resulting in the simultaneous staining of both the calibrator cells and the test specimen.
  • the staining is then measured in the calibrator cells, and this measurement is called the observed value.
  • the slides may sometimes stain a little darker than expected, while at other times the slides may stain a little lighter than expected.
  • This inherent variability is also present in the calibrator cells, and affects their staining, as well.
  • the expected staining intensity of the calibrator cells is known, so if a divergence from the expected is observed, it can be inferred that this divergence represents variability which is not only present in the calibrator cells but in the test specimen as well. This divergence is the difference between the expected value and the observed value.
  • calibrator cells detect variability, but they will also allow for correction of the variability.
  • Image analysis permits the collection of digital images of the calibrator cells and test specimen, and computer algorithms are used to measure the amount of staining. The amount of staining present in the calibrator cells is measured (observed value) and compared to the known expected value. If variability is detected, then the divergence (either positive or negative) from the expected value is calculated. This calculated divergence then becomes a correction factor that is applied to the digital image of the test specimen to remove the variability. This is called normalizing the results to a calibrated standard.
  • kits for IHC Kits for performing IHC are also provided.
  • the kit includes one or more of the disclosed quality control devices.
  • a kit can include at least one of the quality control devices disclosed herein and instructions for using the device.
  • a kit includes at least one quality control device including one or more quality control samples (such as one or more universal positive control cell pellets, antigen-retrieval control cell pellets, endogenous peroxidase control cell pellets, calibrator cell pellets or a combination thereof).
  • the kit also includes reagents for performing IHC (e.g., buffers, detection agents, and the like, which can be in separate containers) and at least one solid support, such as a glass microscope slide, to which the test sample and one or more quality control samples can be attached.
  • reagents for performing IHC e.g., buffers, detection agents, and the like, which can be in separate containers
  • solid support such as a glass microscope slide
  • the IHC assay includes the test sample and one or more control samples, such as a universal positive control, antigen retrieval control, endogenous peroxidase control, or combination thereof as described herein.
  • the IHC staining procedure generally includes five basic steps.
  • a primary antibody specific for the molecule of interest e.g., target antigen
  • a solid support containing test and control samples such as a microscope slide, such that the antibody overlays the control and test samples.
  • the antibody is allowed to incubate with the samples for a sufficient length of time to allow the antigen-antibody reaction to occur.
  • the primary antibody can be any known antibody specific for the antigen of interest (e.g., a mouse monoclonal antibody).
  • the slide is rinsed to remove any unbound primary antibody.
  • a secondary antibody such as an anti-mouse immunoglobulin antibody, is applied to the solid support such that the secondary antibody overlays each of the control and test samples.
  • the secondary antibody will now bind to the primary antibody via a second antigen-antibody reaction. If primary antibody has not bound to the sample, then the secondary antibody will likewise not bind.
  • the secondary antibody is linked to an enzyme, dye, or other detectable label.
  • the secondary antibody can be directly linked to a detectable label, or it can be indirectly linked through a series of steps. In either case, the ultimate result is a physical link between the detectable marker and the secondary antibody. Thus, only if all the steps have been performed correctly will the detectable marker be present. The final step involves detection of the marker.
  • a detectable marker is frequently an enzyme such as peroxidase, which then reacts with a colorless substrate converting it into a colored end-product. The presence of the colored end- product indicates that the molecule of interest is present in the test sample.
  • This example describes the preparation of a universal positive control cell pellet sample.
  • a mouse lymphoma cell line expressing mouse IgG 2A obtained from the American Type Culture Collection (ATCC; TIB- 12) was grown in vitro by placing the cells in a flask containing a nutrient medium composed of RPMI plus 10% bovine calf serum, nonessential amino acids, and L-glutamine. The cells were incubated for three days to allow cell replication. After the desired number of cells was achieved, the cells were harvested, and concentrated into a cell pellet by centrifugation at 100 x g for 10 minutes.
  • the cell pellet was fixed in 95% ethanol for 10 minutes to stop all metabolic activity and to immobilize the antigens within the cells.
  • the fixed cell pellet was then dehydrated in 100% ethanol and cleared in xylene.
  • the cell pellet was dispersed into 200 ⁇ l of molten paraffin at 57 0 C and drawn into the bore of a warmed (57 0 C) glass Pasteur pipette.
  • the pipette contents were then quickly discharged into a recipient paraffin block that had been prepared to accept the cell sample.
  • the recipient paraffin block was prepared by removing a cylindrical core of solid paraffin from the block leaving an empty column measuring approximately 2 mm in diameter.
  • the contents of the Pasteur pipette containing the molten paraffin and cell sample was quickly pipetted into the empty column and solidified at room temperature.
  • the paraffin block, with the embedded cell pellet, was cut into thin slices of approximately 4 microns in thickness.
  • the thin slices were first floated onto a water bath to straighten and were then collected onto a cellophane medium and laid out flat on top of the cellophane medium.
  • the cell pellet section was temporarily attached to the cellophane medium by drying the section, followed by exposing the section to an increased temperature (56°C for five minutes), such that the paraffin melted onto the slide affixing the cell pellet section contained in the paraffin to the cellophane medium.
  • the cell pellet section was then cooled by exposing the section to room temperature (approximately 22°C) such that the paraffin re-solidified.
  • the section was stored at room temperature. This procedure allows a quality control sample to be temporarily attached and subsequently removed from a first surface to adhere to a second surface without damaging the quality control sample.
  • a test sample such as a test tissue or test cell pellet, is prepared in a similar manner as described for the universal positive control cell pellet.
  • the paraffin slices of the universal positive control cell pellet and the test sample are affixed to the same slide and processed using the immunohistochemistry method described above in Example 1.
  • the universal positive control cell pellet expressing mouse immunoglobulin will stain positive any time the IHC procedure has been performed correctly. These positive control cells will stain positive regardless of the staining reaction of the test tissue.
  • the control cells contain mouse immunoglobulin, they will stain positive whenever the secondary antibody of the IHC procedure is applied.
  • the secondary antibody is an anti- mouse immunoglobulin antibody which will always react with the mouse immunoglobulin present on the control cells.
  • a positive staining reaction of the control cells demonstrates that (i) the sample was treated with the secondary antibody and all subsequent steps including any tertiary reactions, enzyme, and substrate were performed correctly; (ii) the protocol from the secondary antibody onward was appropriately followed; and (iii) all reagents from the secondary antibody onward were functional.
  • a positive reaction does not guarantee that the first step (the primary antibody) was performed correctly. Nevertheless, about 80% of the procedure is controlled for by using the universal positive control cell pellet.
  • This example provides an exemplary procedure for preparing a universal positive control cell pellet using the exogenous antigen fluorescein.
  • the cells are prepared similarly to the preparation described in Example 2, with the exception that after the cells are harvested from the tissue culture flasks, they are labeled with fluorescein (Sigma- Aldrich, St. Louis, MO).
  • the universal positive control cell pellet is prepared by chemical conjugation of the fluorescein molecule to the surface of the control cells according to the method of Butcher et al. (J. Immunol. Methods 37(2): 109-21 , 1980). Viable cells are incubated with free fluorescein at a concentration of approximately 10 ⁇ g/ml.
  • the fluorescein is added to the cell growth medium 24 hours prior to harvesting the cells. After harvesting, the cells are separated from unbound fluorescein by centrifugation for 10 minutes at 100 x g.
  • the universal positive control cells and the test tissue can then be processed into paraffin and adhered to a cellophane membrane in a manner similar to the method described in Example 2.
  • the primary antibody reagent (the reagent containing the antibody to the molecule of interest) includes a second antibody to fluorescein.
  • the second antibody does not interfere with the primary antibody to the antigen of interest.
  • the purpose of the second antibody is to react with the universal positive control cells.
  • the test tissue may or may not stain positively, depending on the presence or absence of the antigen of interest, but the universal positive control cells will stain positive by virtue of the reaction of the anti-fluorescein antibody with the fluorescein molecule on the surface of the control cells.
  • a positive stain on the universal positive control cell pellets indicates that (i) a primary antibody was applied; (ii) all steps subsequent to the primary antibody were successfully completed; (iii) all reagents were functional; and (iv) the correct protocol was followed.
  • This example describes the use of a cell line that expresses mouse immunoglobulin as an antigen-retrieval control for an immunohistochemistry assay.
  • the cell pellet is prepared as described above (Example 2) for the universal positive control cell pellet. Briefly, a cell line expressing mouse immunoglobulin, such as a hybridoma cell line, plasmacytoma cell line or a lymphoblastoma cell line, is expanded in vitro to generate the desired number of cells. After expansion, the cells are collected and pelleted. The cell pellet is chemically fixed as part of the preparation process in order to stop all metabolic activity and to preserve the chemical and structural integrity of the cells.
  • the fixative is 10% formalin
  • the expressed mouse immunoglobulin will be chemically modified, resulting cross-linking of the immunoglobulin molecules.
  • Cross-linking of mouse immunoglobulin renders the antigens undetectable by standard IHC staining methods.
  • antigen-retrieval is performed on the chemically modified cells prior to the IHC staining procedure, the mouse immunoglobulin antigens are restored to the native state and can now be stained by the IHC staining procedure.
  • the endogenous peroxidase control cell pellets utilize cells that contain endogenous peroxidase (or pseudo-peroxidase) activity.
  • cells may be chosen from any of a number of suitable cell lines that contain such activity.
  • red blood cell One type of cell containing endogenous peroxidase is a red blood cell. Because red blood cells can be obtained from blood in large quantities, it is possible to harvest red blood cells from a donor animal rather than grow the cells in vitro. However, not all red blood cells are appropriate sources for preparing cell line controls. Mammalian red blood cells do not contain a nucleus, and are therefore, too fragile to embed into paraffin according to the methods described above. In contrast, avian red blood cells are nucleated and are structurally strong enough to be embedded into paraffin.
  • chicken red blood cells are used for the endogenous peroxidase control cell pellet. Chicken red blood cells are readily available through commercial sources.
  • the chicken red blood cells are prepared, embedded into a paraffin block, cut and placed on a microscope slide, as described above in Example 1. During the IHC staining process the slides are exposed to an endogenous peroxidase blocking step. If this procedure is adequate, the endogenous peroxidase control will stain negative. If this procedure is inadequate, the endogenous peroxidase control will stain positive.
  • This example describes an immunohistochemistry assay in which a test sample, universal positive control cell pellet, antigen-retrieval control cell pellet, endogenous peroxidase control cell pellet, or a combination thereof, are attached to the same solid support (such as a glass slide) for processing (see FIG. 4).
  • This example further describes embedding two or more of the control cell pellets into a single paraffin block. This is termed a cell pellet array.
  • the cell pellet array permits all three controls to be placed on the same slide as the test sample and stained at the same time as the test sample. Table II provides an interpretation of staining results that can be obtained from the control cell pellets.
  • the method for preparing a control cell pellet array includes the following steps.
  • Control cell pellets are prepared using standard histological techniques and embedded into a paraffin block as described above.
  • the control cell pellets include a universal positive control cell pellet, an antigen-retrieval control cell pellet and an endogenous peroxidase control cell pellet.
  • the universal positive control cell pellet is prepared from a cell line expressing mouse immunoglobulin and is fixed in ethanol.
  • the antigen-retrieval control cell pellet is prepared from a cell line expressing mouse immunoglobulin and fixed in formalin.
  • the endogenous peroxidase control cell pellet prepared from chicken red blood cells.
  • the control cell pellets can be embedded individually into paraffin blocks or all together in the form of an array.
  • the paraffin block containing one or more of the control cell pellets is cut into a thin paraffin section (e.g. , 2 to 8 microns), which is attached to a microscope slide.
  • a test tissue sample is prepared and embedded into another paraffin block using standard histological methods.
  • the paraffin block containing the test tissue is cut into a thin paraffin section (e.g., 2 to 8 microns) which is attached to the same microscope slide as the control cell pellets. In this way, both the test tissue and the control cell pellets are attached to the same microscope slide.
  • the microscope slide is then stained by an IHC method for an antigen suspected of being present in the test sample.
  • the IHC assay is performed essentially as follows. If needed, antigen retrieval is performed on the slide.
  • the slide is then treated with an endogenous peroxidase inhibitor.
  • a first primary antibody (mouse monoclonal antibody) against the antigen of interest is applied to the slide.
  • An anti-mouse immunoglobulin secondary antibody is then applied, which will bind to the primary antibody and to the universal positive control cell pellet by virtue of the fact that these cells express mouse immunoglobulin.
  • the secondary antibody will also bind to the antigen-retrieval positive control cell pellet if antigen retrieval was performed, but will not bind to the antigen-retrieval positive control cell pellet if antigen retrieval was not performed.
  • the secondary antibody is linked to a peroxidase enzyme, therefore a peroxidase substrate added to the sample to react with the peroxidase enzyme to produce a colored end-product that stains any cells containing the suspected antigen.
  • the peroxidase substrate will simultaneously stain the endogenous peroxidase control cell pellet if the endogenous peroxidase step was inadvertently omitted, or the treatment was inadequate. However, the endogenous peroxidase control cell pellets will not stain if the endogenous peroxidase blocking step was adequate.
  • the staining reaction of the test tissue and control cell pellets is examined under a microscope to evaluate the IHC staining reaction.
  • the slides can be reviewed by conventional observation, or the slides can be analyzed automatically by use of computer-aided image analysis system.
  • Example 7 Preparation of Calibrator Universal Positive Control Cell Pellet This Example describes a method of preparing a calibrator universal positive control pellet.
  • a cell line is grown in vitro by placing cells in a flask or other tissue culture chamber containing a nutrient medium and incubating them for several days so the cells replicate in the flask and increase in number. After the desired number of cells is achieved, the cells are harvested and concentrated into a cell pellet. The cell pellet is fixed, and the fixed cell pellet is then embedded into a paraffin block using standard histological methods. The paraffin block, with the enclosed cell pellet, is then cut into thin slices of approximately 4 microns thick.
  • the slice containing the calibrator cells is then placed on one section ⁇ e.g., end) of a glass microscope slide, and a second paraffin slice containing a test tissue is mounted on a different section ⁇ e.g., the other end) of the glass slide.
  • the general steps of preparing the calibrator positive control cell pellet include (1) growing the cells in vitro, (2) harvesting the cells and concentrating them into a cell pellet, (3) fixing the cell pellet with a fixative, such as 95% ethanol, (4) dehydrating the cell pellet and embedding the cell pellet into a paraffin block, (5) cutting a thin slice off the paraffin block that includes a portion of the cell pellet, (6) allowing the paraffin slice to spread out by floating it on top of a water bath (7) attaching the floating paraffin section containing the calibrator cells onto a microscope slide, (8) attaching a test tissue specimen onto the same microscope slide (but at a different location), (9) staining both the test tissue and the calibrator cells using IHC or another staining procedure, and (10) analyzing the staining results of the test tissue and the calibrator cells by either visual observation under a microscope or by image analysis.
  • a fixative such as 95% ethanol
  • This example describes the use of universal positive control samples prepared in the above Examples.
  • forceps were used to remove the cell pellet from the cellophane membrane and place such pellet onto the second medium.
  • a universal positive control sample was peeled off the cellophane membrane with the aid of forceps, placed onto a glass microscope slide containing the test tissue and permanently affixed to a glass slide next to a test sample.
  • the permanent attachment was achieved by placing the cell pellet into a 10 ⁇ l droplet of water that had been placed onto the glass slide.
  • the slide was then exposed to an elevated temperature (e.g., 57 0 C) to evaporate the water and melt the paraffin. This process permanently attached the universal positive control cell pellet sample onto the glass slide.
  • FIG. 1 shows a microscopic view of a universal positive control cell pellet sample after it has been stained.
  • the staining procedure utilized a mouse monoclonal antibody to vimentin (V9). Slides were first blocked for endogenous peroxidase activity by incubation for five minutes at room temperature with 3% hydrogen peroxide. Slides were then rinsed with phosphate buffered saline and incubated for 10 minutes at room temperature with vimentin antibody (V9). Following incubation with the primary antibody, slides were rinsed in phosphate buffered saline and incubated for 10 minutes at room temperature with the secondary antibody (Envision Plus-HRP, Dako Corp., Carpineria, CA).
  • V9 mouse monoclonal antibody to vimentin
  • FIG. 1 illustrates the individual cells that comprise the cell pellet. The brown color around each cell was indicative of a positive immunohistochemical reaction.
  • FIG. 2 provides a microscopic view (1Ox) of the human tonsil test sample after immunohistological staining for the vimentin antigen. Previous studies have demonstrated that the vimentin antigen is found in various tissue elements contained within tonsil. A positive reaction occurred as indicated by the brown- staining elements.
  • FIG. 3 shows a microscopic view (4Ox) of the universal positive control cell pellet. These control cells contained naturally occurring mouse immunoglobulins which stain positive whenever the immunohistological staining has been completed successfully. Positive staining was indicated by the brown color reaction. Taken together, these data indicate that the immunohistochemical staining procedure was completed successfully and that the observed vimentin staining does in fact represent specific staining, and not background, artifact, or other error in the immunohistochemical staining method.
  • This Example provides an exemplary method for calibrating IHC staining using a calibrator cell pellet. Briefly, a microscope slide that has mounted on it a test sample and a calibrator cell pellet (e.g., see Example 7) is stained such that both the calibrator cells and test specimen are simultaneously stained under identical conditions.
  • a calibrator cell pellet e.g., see Example 7
  • All IHC methods produce the same end result: a positive stain if the antigen of interest was present in the test specimen and a negative stain if the antigen of interest was not present in the test specimen.
  • the calibrator cells stain positive, this is indicative that the IHC method was successful.
  • the calibrator cells act as a positive control.
  • the calibrator cells contain a known level of antigen that provides an expected level of positive staining. Each time the calibrator cells are stained they, should theoretically provide the same expected level of staining.
  • the level of staining is determined using image analysis, which is a process of measuring the amount of staining in a tissue by using a camera (e.g., a CCD camera) to capture an image and then using a computer to analyze the image. Image analysis is more precise in quantifying staining compared to simple visual analysis by the unaided human eye.
  • any deviation from this theoretical limit can be analyzed by image analysis, and a correction factor is calculated. This same correction factor is then applied to the staining intensity measured for the test sample. This process normalizes the test tissue to its internal calibration standard. By normalizing each test sample, the usual intra- and inter-run variability can be eliminated. Not only does this provide more accurate results, but it also allows comparisons of one test sample to another test sample.
  • the calibration is carried out manually.
  • the method can include (1) evaluating the staining results of the calibrator cells by visual observation under a microscope, (2) comparing the observed staining results of the calibrator cells to the expected staining results (for instance, the expected staining results may be in the form of a digital image that displays the expected staining pattern), (3) estimating the deviation from the optimal staining pattern by assigning a score of -4 to +4, wherein 0 represents no deviation and +1 to +4 represent overstaining and -1 to-4 represent understaining, (4) evaluating the staining results of the test sample and assigning it an initial value of 0 to 4 based on its staining intensity, with 0 being negative and 4 being strongly positive, and (5) combining the score from step 3 with the score from step 4 to come up with a corrected score.
  • the combined score cannot be less than 0 or greater than 4.
  • calibration is by image analysis.
  • the method can include evaluating the staining of the calibrator cells by an image analysis method using an algorithm to quantify the observed staining intensity.
  • a common image analysis algorithm assigns a score of 0 - 255 with 0 being the most intensely stained and 255 being negative.
  • the observed staining of the calibrator cells is then compared to the expected staining, and a correction factor is calculated based on the deviation between the observed staining of the calibrator cells and the expected staining.
  • the staining of the test specimen is then evaluated by an image analysis algorithm and a value is assigned (usually between 0 - 255).
  • the correction factor is then applied to the test specimen to normalize the results to the calibrator cells and a new stain value is calculated.
  • test samples that were under- and over-stained were normalized using the calibrator cell pellets.
  • FIGS. 5-8 serial sections of the same tissue were cut from the same paraffin block and mounted on separate microscope slides. Because all three test sections were cut from the same sample, they would be expected to stain with nearly identical results if the same staining protocol is used with each.
  • Calibrator cells were mounted on each slide along with the test specimens. In order to simulate staining variability, slide 1 was under-incubated with the primary antibody in order to produce an under-stain, slide 2 was incubated in the primary antibody for the correct amount of time, and slide 3 was over-incubated with the primary antibody in order to produce an over-stain.
  • the staining variability depicted in FIGS. 5-8 simulates the variability that might be encountered with the IHC method.
  • FIG. 5 shows a comparison of the staining intensity of the calibrator cells on each of the three slides.
  • the histogram beneath each panel shows the calculated staining for the calibrator cells.
  • FIG. 6 shows the staining intensity for each test sample on each of the three slides.
  • the three test samples were stained concurrently with their respective calibrator cell pellets.
  • the lower panels show histograms for the staining of each tissue.
  • FIG. 7 shows the corrected IHC results that have been adjusted using the correction factor derived from the calibrator cell pellet staining. All three test samples show similar staining in the lower (corrected) panels.
  • FIG. 8 shows the adjusted histograms for the test samples. The adjusted final scores for each histogram were 171, 175, and 171, respectively.

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Abstract

L'invention concerne l'immunohistochimie, et en particulier un dispositif de contrôle qualité destiné à une épreuve biologique d'immunohistochimie, ainsi que procédés d'utilisation de ce dispositif. En particulier, l'invention concerne un granulé cellulaire étalonneur et ses procédés d'utilisation en tant que témoin dans une épreuve biologique d'immunohistochimie. Ces granulés cellulaires étalonneurs sont utilisés, dans certains modes de réalisation de l'invention, en tant qu'étalons pour quantifier l'intensité de la coloration immunohistochimique, par exemple pour normaliser cette coloration immunohistochimique afin d'obtenir une coloration étalon, ce qui permet une quantification plus précise de l'intensité de coloration.
PCT/US2008/085841 2007-12-28 2008-12-08 Dispositif cellulaire étalonneur de contrôle qualité destiné à une épreuve biologique d'immunohistochimie et procédés d'utilisation de ce dispositif WO2009085575A2 (fr)

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WO2022233684A1 (fr) * 2021-05-06 2022-11-10 Futamura Chemical Uk Ltd Dispositif et procédé de contrôle de qualité de coloration de tissu
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CN110753846B (zh) * 2017-06-15 2024-03-26 深圳市诺高实验器材有限公司 免疫组织化学抗原成像标尺外推方法
CN113515077A (zh) * 2021-04-23 2021-10-19 重庆德方信息技术有限公司 人体细胞染色过程监控系统及方法
WO2022233684A1 (fr) * 2021-05-06 2022-11-10 Futamura Chemical Uk Ltd Dispositif et procédé de contrôle de qualité de coloration de tissu
GB2606387B (en) * 2021-05-06 2024-08-14 Futamura Chemical Uk Ltd Device
GB2630694A (en) * 2021-05-06 2024-12-04 Futamura Chemical Uk Ltd Device
GB2630694B (en) * 2021-05-06 2025-04-30 Futamura Chemical Uk Ltd Device
CN113820286A (zh) * 2021-09-27 2021-12-21 麦克奥迪（厦门）医疗诊断系统有限公司 用于dna倍体分析系统校准的芯片及校准方法
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CN118376792B (zh) * 2024-04-16 2025-04-15 北京菲诺维康生物科技有限公司 一种检测组织目标蛋白表达结果的校准方法

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