WO2009148885A2

WO2009148885A2 - Hematoxylin staining method to address gradient staining

Info

Publication number: WO2009148885A2
Application number: PCT/US2009/045190
Authority: WO
Inventors: Jonathan Beer; Kurt Reinhardt
Original assignee: Ventana Medical Systems, Inc.
Priority date: 2008-05-30
Filing date: 2009-05-26
Publication date: 2009-12-10
Also published as: WO2009148885A3

Abstract

The present invention is directed to any process and/or composition that substantially reduces or prevents gradient hematoxylin staining of tissue samples. Without being bound by a theory of operation, one possible explanation for observed gradient staining results is the formation of a more reactive staining species produced as a result of a pH change that occurs during the staining process. For an automated system, one disclosed embodiment comprises dispensing a hematoxylin stain onto a tissue sample, and then dispensing a bluing composition to the tissue sample to form a combined hematoxylin stain-bluing composition. The hematoxylin stain-bluing composition is then mixed, such as by agitation, on the slide sufficiently to reduce or eliminate gradient staining. Other approaches also can be employed, either alone or in combination with thoroughly mixing the hematoxylin stain-bluing composition on the slide. For example, the method may comprise treating the sample with process solutions, such as wash solutions, having a pH at or about that of the hematoxylin stain, such as a pH of from about 1 to about 6, more typically from about 2 to about 3.

Description

HEMATOXYLIN STAINING METHOD TO ADDRESS GRADIENT STAINING

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority to and claims the benefit of U.S. Provisional Patent Application No. 61/130,442 filed on May 30, 2008, and is incorporated herein by reference in its entirety.

FIELD

The present invention concerns embodiments of a method and/or compositions that reduce or substantially eliminate gradient staining that may occur when staining a sample using hematoxylin staining compositions.

BACKGROUND

Compositions comprising hematoxylin and hematein (also haematoxylin and haemetein), the chemical structures of which are provided below, are commonly used in pathology (the microscopic examination of fixed cytology specimens, i.e. individual cells in a smear or cell block) and histology (microscopic examination of cell aggregates that form a structure with a specific function). For example, hematoxylin and hematein are often used to stain cell nuclei prior to microscopic examination. Hematoxylin oxidizes to form hematein, which has a rich blue-purple color.

Hematoxylin

Hematein

Staining makes normally transparent cells colored, which facilitates analysis. Hematoxylin staining can be accomplished either manually using an immersion (dip and dunk) technique or by using automated systems, such as the Symphony^® automated system provided by Ventana Medical Systems, Inc. The staining processes generally involve: (a) removing paraffin from a specimen affixed to a microscope slide and hydrating the specimen by soaking in water; (b) applying hematoxylin to stain cell nuclei; (c) removing excess hematoxylin by rinsing with water; (d) contacting the slide with a concentrated solution having a pH above 5.0 to turn the hematoxylin blue [bluing solution]; and (e) removing the bluing solution by rinsing with water. For manual processes, hematoxylin staining typically is performed using aqueous staining baths with the slides oriented vertically. Rinsing occurs in a second bath, again with the slides held vertically. Alternatively, and particularly for automated systems, slides are positioned horizontally and rinsed from a first end proximal to a wash composition application point to a second distal end of the slide relative to the wash solution application point.

Recently, gradient staining has been observed, particularly when samples are stained using darker stain compositions comprising relatively high concentrations of hematein. "Gradient staining" refers to a staining pattern where samples stain from a region of relatively light staining, and which increases in staining intensity towards a region of relatively darker staining. With reference to a particular gradient staining pattern that has been observed, "gradient staining" refers to a staining pattern whereby the sample is stained darker distally from a point where a wash composition is applied to the slide than occurs proximal to the point where the wash composition is applied. Compare FIG. 1 to FIG. 2. For example, gradient staining has been observed on small biopsies where serial slide samples are taken from the

- ? - same tissue.

One factor considered indicative of cancerous tissue is darker stained regions, as darker staining indicates a higher density of nuclei. Staining protocols that induce darker stained regions on certain samples create the possibility for false positives. A preferred process therefore would reduce or substantially eliminate gradient staining that may occur when using hematoxylin/hematein staining compositions.

SUMMARY Disclosed embodiments of the present invention address differential staining results that occur when using hematoxylin staining protocols. Without being bound by a theory of operation, one possible explanation for these observed gradient staining results is the formation of a more reactive staining species, such as may be produced as a result of a pH change that occurs during the staining process. Thus, the present invention is particularly directed to any process and/or composition that substantially reduces or prevents gradient hematoxylin staining of the tissue sample. The method may be an automated staining method or a manual staining method.

For an automated system, one disclosed embodiment comprises first dispensing a hematoxylin stain onto a tissue sample. A bluing composition is then applied to the tissue sample to form a combined hematoxylin stain-bluing composition. The hematoxylin stain-bluing composition is then mixed, such as by agitation, on the slide sufficiently to reduce or eliminate gradient staining. Any of various hematoxylin staining compositions can be used. Solely by way of example, and without limitation, a first suitable hematoxylin stain comprises deionized water, ethylene glycol, hematoxylin dye, sodium iodate, aluminum sulfate hydrate and glacial acetic acid. A second suitable hematoxylin stain comprises deionized water, ethylene glycol, hematoxylin dye, sodium iodate, aluminum sulfate, hydroquinone and β-cyclodextrin hydrate. Automated systems also are capable of performing at least one additional staining protocol, such as cytoplasmic staining. Automated systems typically are at least partially, if not substantially entirely, under computer control. Because automated systems typically are at least partially computer controlled, certain embodiments of the present invention also concern one or more tangible computer-readable media that stores computer- executable instructions for causing a computer to perform disclosed embodiments of the method. For example, a working embodiment that is controlled by a computer comprises first deparaffinizing the sample, if required. A hematoxylin stain composition is then applied to the sample, followed by addition of a bluing reagent or composition. The automated system then air mixes the hematoxylin stain and the bluing reagent on the slide by dispensing plural air pulses onto the slide, each air pulse having a duration effective to mix the reagents, such as from about 20 to about 150, more typically from about 50 to about 100 milliseconds. The air pulse also is applied at a pressure effective to mix the reagents. This pressure may vary, depending on various factors such as distance of application from the slide surface, but typically is from about 5 to about 20 psi. For certain disclosed working embodiments the air pressure was applied at from about 14 to about 16 psi, and most typically involved dispensing at least one air pulse at about 15 psi. Thoroughly mixing the hematoxylin stain-bluing composition is one method for addressing gradient staining. However, other approaches also can be employed, either alone or in combination with thoroughly mixing the hematoxylin stain-bluing composition on the slide. For example, the method may comprise dispensing a hematoxylin stain onto a sample, and incubating the sample for a period of time effective to effectively stain the sample. The sample is then washed with a wash solution having a pH at or about that of the hematoxylin stain. Any hematoxylin stain composition can be used, and such compositions have pH values that range from about 1 to about 6, more typically from about 2 to about 5, and even more typically from about 2 to about 3. The method also can comprise treating the sample with a bluing solution having a pH at or about that of the hematoxylin stain. For a manual staining method, the sample is immersed at least once in an aqueous solution comprising 0.1 to 2% hematoxylin for a period of time effective to stain the sample. The sample is then washed with a wash solution buffered to a pH of from about 1 to about 6, more typically from about 2 to about 5, and even more typically from about 2 to about 3.

Accordingly, disclosed embodiments also concern hematoxylin stain wash compositions having a pH substantially equal to or about that of the hematoxylin stain composition, such as a pH of from about 1 to about 6, more typically from about 2 to about 5, and even more typically from about 2 to about 3.

The foregoing and other objects, features, and advantages of the invention will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photograph of GI tissue stained on a slide.

FIG. 2 is a photograph of GI tissue stained on the same slide as FIG. 1 but having darker stains than that of FIG. 1 illustrating the staining gradient that has been observed when using hematoxylin/hematein staining compositions.

FIG. 3 is a schematic illustration of a stained slide illustrating the gradient staining results that are observed with certain staining protocols.

FIG. 4 is a photograph of GI tissue stained on a slide illustrating the lighter stained tissue on the left versus the darker stained tissue on the right side of the slide distal to the initial application of wash solution.

FIG. 5 illustrates the same tissue as that of FIG. 4 but stained using embodiments of the present invention to mitigate gradient staining results.

FIG. 6 is a flow chart of computer-controlled process steps prior to implementation of process steps useful for mitigating gradient staining.

FIG. 7 is a flow chart of computer-controlled process steps useful for mitigating gradient staining according to certain disclosed embodiments of the present invention.

DETAILED DESCRIPTION

I. Terms

Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found in the technical literature, such as Benjamin Lewin, Genes VII, published by Oxford University Press, 2000 (ISBN 019879276X); Kendrew et al. (eds.), The

Encyclopedia of Molecular Biology, published by Blackwell Publishers, 1994 (ISBN 0632021829); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by Wiley, John & Sons, Inc., 1995 (ISBN 0471186341); and other similar references.

As used herein, the singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Also, as used herein, the term "comprises" means "includes." Hence "comprising A or B" means including A, B, or A and B. It is further to be understood that all amino acid sizes, and all molecular weight or molecular mass values, given for polypeptides or other compounds are approximate, and are provided for description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.

In order to facilitate review of the various examples of this disclosure, the following explanations of specific terms are provided:

Animal: Living multi-cellular vertebrate organisms, a category that includes, for example, mammals and birds.

Antibody: "Antibody" collectively refers to immunoglobulins or immunoglobulin- like molecules (including by way of example and without limitation, IgA, IgD, IgE, IgG and IgM, combinations thereof, and similar molecules produced during an immune response in any vertebrate, for example, in mammals such as humans, goats, rabbits and mice), as well as non-mammalian species, such as shark immunoglobulins. "Antibody" also includes antibody fragments that specifically bind to a molecule of interest (or a group of highly similar molecules of interest) to the substantial exclusion of binding to other molecules (for example, antibodies and antibody fragments that have a binding constant for the molecule of interest that is at least 10³ M^"1 greater, at least 10⁴ M^"1 greater or at least 10⁵ M^"1 greater than a binding constant for other molecules in a biological sample.

More particularly, "antibody" refers to a polypeptide ligand comprising at least a light chain or heavy chain immunoglobulin variable region which specifically recognizes and binds an epitope of an antigen. Antibodies are composed of a heavy and a light chain, each of which has a variable region, termed the variable heavy (V_H) region and the variable light (V_L) region. Together, the V_H region and the V_L region are responsible for binding the antigen recognized by the antibody.

This includes intact immunoglobulins and the variants and portions of them well known in the art. Antibody fragments include proteolytic antibody fragments [such as F(ab')₂ fragments, Fab' fragments, Fab'-SH fragments and Fab fragments as are known in the art], recombinant antibody fragments (such as sFv fragments, dsFv fragments, bispecific sFv fragments, bispecific dsFv fragments, F(ab)'₂ fragments, single chain Fv proteins ("scFv"), disulfide stabilized Fv proteins ("dsFv"), diabodies, and triabodies (as are known in the art), and camelid antibodies (see, for example, U.S. Patent Nos. 6,015,695; 6,005,079- 5,874,541 ; 5,840,526; 5,800,988; and 5,759,808). An 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 genetically engineered 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, J., Immunology, 3^rd Ed., W.H. Freeman & Co., New York, 1997.

Typically, a naturally occurring immunoglobulin has heavy (H) chains and light (L) chains interconnected by disulfide bonds. There are two types of light chain, lambda (λ) and kappa (K). There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE.

Each heavy and light chain contains a constant region and a variable region, (the regions are also known as "domains"). In combination, the heavy and the light chain variable regions specifically bind the antigen. Light and heavy chain variable regions contain a "framework" region interrupted by three hypervariable regions, also called "complementarity-determining regions" or "CDRs". The extent of the framework region and CDRs have been defined (see, Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991, which is hereby incorporated by reference). The Kabat database is now maintained online. The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework region of an antibody, that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs in three-dimensional space.

The CDRs are primarily responsible for binding to an epitope of an antigen. The CDRs of each chain are typically referred to as CDRl, CDR2, and CDR3, numbered sequentially starting from the N-terminus, and are also typically identified by the chain in which the particular CDR is located. Thus, a V_H CDR3 is located in the variable domain of the heavy chain of the antibody in which it is found, whereas a V_L CDRl is the CDRl from the variable domain of the light chain of the antibody in which it is found. An antibody that binds RET will have a specific V_H region and the V_L region sequence, and thus specific CDR sequences. Antibodies with different specificities (i.e. different combining sites for different antigens) have different CDRs. Although it is the CDRs that vary from antibody to antibody, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. These positions within the CDRs are called specificity determining residues (SDRs).

Antigen: A compound, composition, or substance that may be specifically bound by the products of specific humoral or cellular immunity, such as an antibody molecule or T-cell receptor. Antigens can be any type of molecule including, for example, haptens, simple intermediary metabolites, sugars (e.g., oligosaccharides), lipids, and hormones as well as macromolecules such as complex carbohydrates

(e.g., polysaccharides), phospholipids, and proteins. Common categories of antigens include, but are not limited to, viral antigens, bacterial antigens, fungal antigens, protozoa and other parasitic antigens, tumor antigens, antigens involved in autoimmune disease, allergy and graft rejection, toxins, and other miscellaneous antigens. In one example, an antigen is a Bacillus antigen, such as γPGA.

Antioxidant: An atom or molecule that has a greater oxidation potential than a second atom or molecule, such that the antioxidant is preferentially oxidized instead of the second atom or molecule. For example, an antioxidant can have a greater oxidation potential than hematein, and thus help prevent oxidation of hematein to oxyhematein. Furthermore, an antioxidant also can function as a reducing agent, for example, a reducing agent that converts oxyhematein back to hematein. Antioxidants can be present in the disclosed compositions at concentrations ranging from about about 1 mM to about IM, for example, from about 5 mM to about 500 mM, such as from about 50 mM to about 150 mM.

Bluing, Bluing Reagent, Composition or Solution: Nuclei stained with hematoxylin often are at first stained the purplish color of the acid dye. Changing the staining color to blue provides better contrast with counterstains, such as the usual red counterstains. When a staining endpoint has been reached by either progressive or regressive methods, nuclear color can be changed in one of two ways. The slides may be contacted with a bluing solution, such as a weakly alkaline solution including, without limitation, ammonia water or dilute sodium carbonate. Depending on the geographic area and the local method of water treatment, tap water tends to be slightly acid, with a pH in the range of 6.0 - 6.8. However, this is considerably more alkaline than the pH of most alum hematoxylins (2.6 - 2.9), so bluing results. The tap water wash has the added advantage that it washes out any excess alum, giving a crisper nuclear stain and preventing fading during storage. Computer-readable media: Computer readable media or CRM refers to any device or system (e.g., machine or tool) for storing and providing information (e.g., instructions, etc.) to a computer processor. Examples of computer-readable media include, but are not limited to, a storage disk, a floppy disk, RAM, ROM, computer chips, digital video disc (DVDs), compact discs (CDs), hard disk drives (HDD), flash memory, and magnetic tape. A computer processor or central processing unit (CPU) are used interchangeably and refer to any hardware and software combination device that is able to read computer readable-media and perform a set of steps according to a program. An exemplary processor is a programmable digital microprocessor such as that available in an instrument that is used in performing automated staining of tissue samples as described herein, or it may be a microprocessor as found in a mainframe computer, a server, or a personal computer. For example, one or more steps or processes as exemplified in FIG. 7 for staining tissue for subsequent microscopic examination are provided by one or more tangible computer-readable media comprising instructions for performing the one or more steps or processes for automated methods for staining a tissue sample for microscopic examination. 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.

Hematoxylin, Hematoxylin Stain or Hematoxylin Composition: Genetically refers both to compositions formed by dissolving hematein (the oxidation product of hematoxylin) directly into a solvent and to compositions formed by dissolving hematoxylin in a solvent and allowing or promoting oxidation of the hematoxylin to hematein. Although it is more typical to prepare the disclosed compositions by dissolving hematoxylin in a solvent and converting the hematoxylin to hematein (either completely or partially) by natural oxidation through contact with air or accelerated chemical oxidation, such as by using an iodate, these terms also refer compositions prepared by directly dissolving hematein in solvent. Thus, in some embodiments, a "hematoxylin composition" will include, at least initially, little or no hematoxylin, and consist primarily of hematein. Host Compound: An organic or inorganic molecule, complex or material having an inner cavity portion or groove portion, and more particularly, to a molecule having an inner cavity portion or groove portion that can accommodate at least a portion of a hematein or other dye molecule. Host compounds include polysaccharides such as amyloses, cyclodextrins and other cyclic or helical compounds containing a plurality of aldose rings, for example, compounds formed through 1,4 and 1,6 bonding of monosaccharides (such as glucose, fructose, and galactose) and disaccharides (such as saccharose, maltose, and lactose). Other host compounds include cryptands, cryptophanes, cavitands, crown ethers, dendrimers, nanotubes, calixarenes, valinomycins, and nigericins. In particular embodiments, a host compound can be a cyclodextrin or cyclodextrin derivative, and more particularly, a host compound can be a cyclodextrin or cyclodextrin derivative exhibiting water solubility at 25⁰C or greater than 5 mg/mL, such as greater than 20 mg/mL, greater than 100 mg/mL, or even greater than 500 mg/mL. In other particular embodiments, a host compound can be α-amylose, β-amylose or V- amylose. Host compounds can be included at concentrations ranging from about 1 mM to about IM, for example, from about 5 mM to about 500 mM, such as from about 5 mM to about 25 mM.

Host compounds can include cyclodextrin derivatives, amylose derivatives, cryptand derivatives, cryptophane derivatives, cavitand derivatives, crown ether derivatives, dendrimer derivatives, nanotube derivatives, calixarene derivatives, valinomycin derivatives, and nigericin derivatives modified with one or more substituents. For example, host compounds include amylose derivatives and cyclodextrin derivatives, wherein one or more of the hydroxyl groups or the hydrogen atoms of the hydroxyl groups of their constituent aldose rings are replaced with substituents. Examples of substituents include acyl groups (such as acetyl groups), alkyl groups, aryl groups, tosyl groups, mesyl groups, amino groups (including primary, secondary, tertiary and quaternary amino groups), halo groups (- F, -Cl, -Br and -I), nitro groups, phosphorous-containing groups (such as phosphate and alkylphosphate groups), sulfur-containing groups (such as sulfate and sulfate ester groups), bridging groups, (that, for example, connect two or more hydroxyl positions on a cyclodextrin ring or connect two or more host compounds), aldehyde groups, ketone groups, oxime groups, carboxylic acid groups and their derivatives, carbonate and carbamate groups, silicon-containing groups, boron-containing groups, tin-containing groups, and hydroxyalkyl groups (such as hydroxyethyl groups and hydroxypropyl groups).

Particular examples of cyclodextrins include α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, and δ-cyclodextrin, and derivatives of each of these classes of cyclodextrins. Particular examples of cyclodextrin derivatives, include hydroxypropylated α-cyclodextrin, hydroxypropylated β-cyclodextrin, hydroxypropylated γ-cyclodextrin, hydroxyethylated α-cyclodextrin, hydroxyethylated β-cyclodextrin, hydroxyethylated γ-cyclodextrin, hydroxyisopropylated α-cyclodextrin, hydroxyisopropylated β-cyclodextrin, hydroxyisopropylated γ-cyclodextrin, carboxymethylated α-cyclodextrin, carboxymethylated β-cyclodextrin, carboxymethylated γ-cyclodextrin, carboxyethylated α-cyclodextrin, carboxyethylated β-cyclodextrin, carboxyethylated γ-cyclodextrin, octyl succinated-α-cyclodextrin, octyl succinated- β-cyclodextrin, octyl succinated-γ-cyclodextrin, acetylated-α-cyclodextrin, acetylated -β- cyclodextrin, acetylated -γ-cyclodextrin, sulfated-α-cyclodextrin, sulfated-β- cyclodextrin and sulfated-γ-cyclodextrin. Other particular examples of cyclodextrins derivatives include the following β-cyclodextrin derivatives: 2,3- dimethyl-6-aminomethyl-β-cyclodextrin, 6- Azido- β-cyclodextrin, 6-Bromo-β- cyclodextrin, 6A,6B-dibromo-β-cyclodextrin, 6A,6B-diiodo-β-cyclodextrin, 6-0- MaI tosyl- β-cyclodextrin, 6-Iodo- β-cyclodextrin, 6-Tosyl- β-cyclodextrin, Peracetyl- maltosyl- β-cyclodextrin, 6-z-butyldimethylsilyl-β-cyclodextrin, 2,3-diacetyl-6- butyldimethylsilyl- β-cyclodextrin, 2, 6-dibutyl-3 -acetyl- β-cyclodextrin, 2,6-dibutyl- β-cyclodextrin, 2,6-f-butyl-dimethylsilyl-β-cyclodextrin, and 2,6-di-O-methyl-3- allyl- β-cyclodextrin. A variety of cyclodextrins and cyclodextrin derivatives can be obtained commercially, for example, from CTD, Inc. (High Springs, FL), or they can be synthesized according to procedures outlined in the scientific literature, for example, in "Synthesis of Chemically Modified Cyclodextrins," Croft and Bartsch, Tetrahedron, 39: 1417-1474, 1983.

Mammal: This term includes both human and non-human mammals. Similarly, the term "subject" includes both human and veterinary subjects. Molecule of Interest or Target: A molecule for which the presence, location and/or concentration is to be determined. Examples of molecules of interest include proteins tagged with haptens.

Mordant: An ionic metal species with which a dye (such as hematein) can form a complex (such as a cationic complex) that serves to bind the dye (such as hematein) to particular cellular components such as nuclear DNA, myelin, elastic and collagen fibers, muscle striations and mitochondria. Examples of mordants include aluminum (for example, in the form of an alum such as aluminum sulphate, aluminum potassium sulphate or aluminum ammonium sulphate), iron, tungsten, zirconium, bismuth, molybdenum (phosphomolybdic acid or molybdic acid), vanadium (vanadate).

Neoplasia and Tumor: The process of abnormal and uncontrolled growth of cells. Neoplasia is one example of a proliferative disorder. The product of neoplasia is a neoplasm (a tumor), which is an abnormal growth of tissue that results from excessive cell division. A tumor that does not metastasize is referred to as "benign." A tumor that invades the surrounding tissue and/or can metastasize is referred to as "malignant." Examples of hematological tumors include leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia and myeloblasts, promyelocytic, myelomonocytic, monocytic and erythroleukemia), chronic leukemias (such as chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and high grade forms), multiple myeloma,

Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia.

Examples of solid tumors, such as sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder carcinoma, and CNS tumors (such as a glioma, astrocytoma, medulloblastoma, craniopharyogioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma and retinoblastoma).

Oxidant: An atom or molecule having a greater reduction potential than a second molecule, for example, a greater reduction potential than hematoxylin such that it will react with and oxidize hematoxylin to hematein. Oxidants include naturally occurring molecular oxygen in the atmosphere that diffuses to and oxidizes hematoxylin and a "chemical oxidant" that is actively combined with hematoxylin (typically in solution) to convert at least a portion of the hematoxylin to hematein. Examples of useful chemical oxidants include one or more of an iodate salt (such as sodium iodate and potassium iodate), mercuric oxide, a permanganate salt (such as potassium permanganate), a periodate salt (such as sodium periodate and potassium periodate), and a peroxide (such as hydrogen peroxide). In particular embodiments, the chemical oxidant comprises sodium iodate.

Papanicolaou, or Pap, Stain: Refers to a multichromatic histological staining technique developed by George Papanikolaou that is used to differentiate cells in smear preparations of various bodily secretions, including gynecological smears, sputum, brushings, washings, urine, cerebrospinal fluid, abdominal fluid, pleural fluid, synovial fluid, seminal fluid, needle aspiration material, tumor touch samples, or other materials containing cells. Pap staining typically involves using five dyes: A nuclear hematoxylin stain for staining cell nuclei; first OG-6 counterstain (-6 refers to using phosphotungstic acid; other variants are OG-5 and OG-8); a second EA Eosin Azure counterstain, comprising three dyes, where the number denotes the proportion of the dyes, e.g. EA-36, EA-50, EA-65; Eosin Y stain to stain superficial epithelial squamous cells, nucleoli, cilia and red blood cells; light green SF yellowish to stain the cytoplasm of all other cells (this dye is now quite expensive and difficult to obtain, and therefore Fast Green FCF dye can be used instead); and optionally Bismarck Brown Y stain. The stained specimen displays hues from the entire spectrum: red, orange, yellow, green, blue, and violet. Cell nuclei are crisp blue to black. Cells with high content of keratin are yellow, glycogen stains yellow as well. Superficial cells are orange to pink, and intermediate and parabasal cells are turquoise green to blue. Metaplastic cells often stain both green and pink. Additional information concerning Papanikolaou stains is provided by U.S. Patent No. 7,186,522, which is incorporated herein by reference. Polypeptide: A polymer in which the monomers are amino acid residues which are joined together through amide bonds. When the amino acids are alpha- amino acids, either the L-optical isomer or the D-optical isomer can be used. The terms "polypeptide" or "protein" as used herein are intended to encompass any amino acid sequence and include modified sequences such as glycoproteins. The term "polypeptide" is specifically intended to cover naturally occurring proteins, as well as those which are recombinantly or synthetically produced. The term "residue" or "amino acid residue" includes reference to an amino acid that is incorporated into a protein, polypeptide, or peptide.

Progressive Staining: Refers to contacting a sample just long enough to reach the proper endpoint, which may require examining the slides at several different intervals to determine when staining is dark enough but not too dark.

Protein: A molecule, particularly a polypeptide, comprised of amino acids. Regressive Staining: Refers to deliberately overstaining a sample, and then destaining (differentiating) until the proper endpoint is reached. Regressive hematoxylins typically are more concentrated than progressive hematoxylins and many can achieve overstaining in a matter of less than a minute, while differentiation (removal of excess stain) requires only a few seconds.

Sample: The term "sample" refers to any liquid, semi-solid or solid substance (or material) in or on which a target can be present. In particular, a sample can be a biological sample or a sample obtained from a biological material. Examples of biological samples include tissue samples and cytology samples, with more particular examples including, peripheral blood, urine, saliva, tissue biopsy, surgical specimen, amniocentesis samples and autopsy material.

Subject: Includes both human and veterinary subjects, for example, humans, non-human primates, dogs, cats, horses, and cows. Target: Any molecule for which the presence, location and/or concentration is or can be determined. Examples of target molecules include proteins and haptens, such as haptens covalently bonded to proteins. Target molecules are typically detected using one or more conjugates of a specific binding molecule and a detectable label.

II. Hematoxylin Staining A. Generally

Hematein is considered the active ingredient in hematoxylin staining compositions. Hematoxylin typically is oxidized to hematein in situ using a strong oxidant, such as sodium iodate. Hematein exhibits indicator-like properties, being blue and less soluble in aqueous alkaline conditions, and red and more soluble in alcoholic acidic conditions. To ensure saturation of chemical binding sites, the stain may be applied longer than necessary, resulting in overstaining of the tissues with much non-specific background coloration. This undesirable coloration is selectively removed by controlled leaching in an alcoholic acidic solution, (acid alcohol), the process being termed "differentiation". Differentiation is arrested by returning to an alkaline environment, whereupon the hematein takes on a blue hue, a process referred to as "bluing". The hematein is generally useful for visualizing cell nuclei. With reference to exemplary process steps used for hematoxylin staining, an aqueous solution of hematoxylin is applied to the sample, such as by immersion or dispensing the solution onto the sample. This step can be conducted in one stage or in plural stages. The sample is incubated with the hematoxylin stain until staining is complete, which generally requires from about 10 seconds up to at least about an hour, more typically from about 45 seconds up to about 4 minutes. Hematoxylin stain optionally may be applied to the specimen a second or more times. The sample optionally is rinsed with a wash solution, such as deionized water. A staining reaction accelerator, such as a solution of lithium carbonate, also can be applied to the sample. After these steps, the sample is fixed and the cell nuclei are stained with a bluish-purple hue.

If desired, the sample can also be counterstained using any of the well known differentiating stains including, for example, eosin, a fuchsin such as rosaniline, pararosaniline, magenta II, magenta III or acid fuchsin; picric acid or the like. Three common forms of eosin include eosin Yellowish (tetrabromofluorescein, disodium salt CI 45380), eosin Bluish (the dinitro- dibromo-derivative CI 45400), and eosin Alcohol Soluble (the ethyl derivative CI 45386). Traditionally, Eosin Y — Light Green/Fast Green is prepared as a mixture of three stains, Eosin Y — Light Green SF and/or Fast Green FCF, and Bismarck Brown. Exemplary mixtures include EA36, EA50, and EA65. Eosin Y produces a pink color in nucleoli, cilia, red blood cells and the cytoplasm of mature squamous cells. Light Green SF Yellowish and Fast Green FCF produce a blue/green color in the cytoplasm of metabolically active parabasal squamous cells, intermediate squamous cells and columnar cells. Bismarck Brown does not produce a useable staining pattern in pap staining, and can be excluded in variations of the method. Substitutes for Light Green SF Yellowish may also be used, e.g. Fast Green FCF. Cytoplasmic counterstains can also be a mixture of OG and Eosin Y — Light Green/Fast Green resulting in a single solution for use as a counterstain.

General steps used for automated hematoxylin staining protocols, such as used on the Symphony^® platform provided by Ventana Medical Systems, Inc., include deparaffinization, followed by a deparaffinization rinse step. The sample is then prepared for staining, such as by the addition of deionized water. A hematoxylin stain composition is then applied, followed by an incubation period. The sample is then rinsed. The sample is then treated with a bluing composition. Once the bluing step has been completed, a bluing rinse step is performed to remove bluing composition. These steps typically are sufficient to complete the staining process using the hematoxylin-hematein staining composition.

B. Hematoxylin Staining Compositions

Various embodiments of hematoxylin staining compositions can be used, and the present invention is directed to all such compositions. For example, the hematoxylin can be any of the usual forms including, but not limited to, Harris' Hematoxylin, Mayor's Hacmalum, Erlich's Hematoxylin, and alum hematoxylin. Solely by way of example, and without limitation, two hematoxylin staining compositions that are used by Ventana Medical Systems, Inc., the assignee of the present invention, particularly with the Symphony^® automated platform, are provided below.

Table 1 - Li ht Stain

The light stain has a pH of from about 2.1 to about 2.4. Table 2 - Dark Stain

The dark stain has a pH of from about 2.4 to about 2.7. Hematoxylin compositions also can be stabilized compositions, such as disclosed in U.S. Patent Application No. 12/048,749, which is incorporated herein by reference. Briefly, one disclosed embodiment of such a composition includes hematoxylin, a solvent, an amount of a chemical oxidant sufficient to convert at least a portion of the hematoxylin to hematein, a mordant and either or both of a host compound and an antioxidant. In particular embodiments, the composition includes both a host compound and an antioxidant. In even more particular embodiments, the composition includes two or more different antioxidants such as two or more water- soluble antioxidants. In other even more particular embodiments, the composition includes one or more host compounds and one or more antioxidants. The host compound of various embodiments can be one or more of an amylose, a cyclodextrin, a cryptand, a cryptophane, a cavitand, a crown ether, a dendrimer, a nanotube, a calixarene, a valinomycin, and a nigericin. In more particular embodiments, the host compound is one or more of a cyclodextrin or a cyclodextrin derivative, and more particularly one or more of β-cyclodextrin and a β-cyclodextrin derivative. In other more particular embodiments, the host compound can have a water solubility of greater than 100 mg/mL at 25°C.

In some embodiments, the solvent is an aqueous solvent and the antioxidant is a water-soluble antioxidant. Examples of water soluble antioxidants include hydroquinones; n-alkyl gallates (such as n-propyl, n-octyl, and n-dodecyl gallates); reducible sugars such as sorbitol and mannitol; benzoates and hydroxybenzoates; sulfites and metabisulfites; certain acids such as citric acid, tartaric acid, lactic acid, erythorbic acid ascorbic acid, uric acid, tannic acid, and salts of such acids (such as Mg²⁺, NH₄ ⁺, Na⁺, K⁺ and Ca²⁺ salts); chelators such as EDTA that remove metals that function as oxidants; and choral hydrate. In particular embodiments, the water soluble antioxidant includes one or more of hydroquinone and n-propyl gallate.

Various solvents can be utilized for the composition, but typically the solvent comprises one or more of water, a lower alkanol such as ethanol, and a polyol. In particular embodiments, the solvent comprises an aqueous solvent wherein the aqueous solvent comprises water and a polyol. Particular examples of useful polyols include glycerol, ethylene glycol, propylene glycol, poly (ethylene glycol), and poly (propylene glycol). Aqueous solvent compositions typically will comprise 5-45% by volume of one or more of ethylene glycol and propylene glycol, and more typically 10-30% by volume of one or more of ethylene glycol and propylene glycol. The amount of chemical oxidant utilized in some embodiments of the composition can be sufficient to completely (such as substantially quantitatively) oxidize the hematoxylin to hematein, or sufficient only to partially oxidize the hematoxylin to hematein. In particular embodiments, more than half of the hematoxylin is oxidized to hematein by the chemical oxidant, and in others, less than half of the hematoxylin is oxidized to hematein by the chemical oxidant. For example, between 1% and 50% of the hematoxylin can be oxidized to hematein by the chemical oxidant, but more typically, between about 10% and about 30% of the hematoxylin is oxidized to hematein by the chemical oxidant. In particular examples, the molar ratio of hematoxylin to oxidant used in the composition is between 6: 1 and 1 :1. It should be understood that although the chemical oxidant is considered part of the composition, it is converted to its reduction products upon reaction with the hematoxylin, which reduction products will remain in the composition.

The mordant of the composition can be any mordant such as one or more of an aluminum mordant, an iron mordant, a bismuth mordant, a copper mordant, a molybdenum mordant, a vanadium mordant, and a zirconium mordant. In some embodiments, the mordant comprises an alum, and in more particular embodiments, the mordant comprises aluminum sulphate. The mordant can be present in the composition at a concentration greater than the concentration of the hematein in the composition (determinable by refractometry, thin-layer chromatography or spectroscopy), or it can be present in the composition at a concentration less than the concentration of the hematein in the composition. Alternatively, in some embodiments, the molar ratio of hematoxylin to mordant in the composition is between 2: 1 and 1 : 100, and in particular embodiments, the molar ratio of hematoxylin to mordant in the composition is between 1 :5 and 1:20.

In some embodiments, the composition further includes an acid such as acetic acid. In other embodiments, no acid is added, and the absence of the acid surprisingly still provides a stabilized and effective hematoxylin composition. In other embodiments, the composition further includes a buffer to control pH, for example, a buffer to control the pH near a pH between 1 and 4, such as a pH near 2.5. In some particular embodiments, a disclosed composition comprises a mixture of water and ethylene glycol as the solvent, sodium iodate as the oxidant, aluminum sulphate as the mordant, and β-cyclodextrin or a derivative thereof as the host compound. One or more water soluble antioxidants such as hydroquinone and n-propyl gallate also can be included in such particular embodiments. In even more particular embodiments, the mixture of water and ethylene glycol comprises from 10-40% by volume ethylene glycol and from 60-90% water.

In addition to the hematoxylin compositions discussed above, additional examples include Anderson's, Apathy's, Baker's Bennett's, Bohmer's, Bosma's, Bullard's, Carazzi's, Cole's, Debiden's, de Groot's, Delafield's, Duval's, Ehrlich's, Friedlander' s, Gadsdon's, Gage's, Galigher's, Garvey's, Gill's, Graham's, Hamilton's, Harris', Harris & Power's, Haug's, Horneyold's, Kleinenberg's, Krutsay's, Langeron's, Launoy's, Lee's, Lillie's, Lugol's, McLachlan's, Mallory's, Mann's, Martinotti's, Masson's, Mayer's, Mitchell's, Molnar's, Papamiltiades', Pusey's, Rawitz', Reddy's, Sass', Schmorl's, Slidders', Unna's, Watson's, and Weigert & Wright's. Particular examples of iron- mordanted hematoxylin stains include Anderson's, Cretin's, Faure's, Goldman's, Hansen's, Heidenhain's, Janssen's, Kefalas', Krajian's, Krutsay's, La Manna's, Lillie's, Lillie & Earle's, Masson's, More & Bassal's, Murray's, Paquin & Goddard's, Regaud's, Rozas', Seidelin's, Thomas', Weigert's, and Yasvoyn's. A bismuth-mordanted hematoxylin is Roach & Smith's. Copper- mordanted hematoxylins include Bensley's, Cook's and Faure's. A molybdenum-mordanted hematoxylin is Held' s. Vanadium- mordanted hematoxylins include Hedenhain's, and Smith's. A zirconium- mordanted hematoxylin is McNulty & Smith's. Formulas and methods of making and using such mordanted hematoxylin solutions can be found, for example, in the StainsFile (an internet resource for histotechnologists maintained by Bryan Llewellyn); Kiernan, "Histological and Histochemical methods: Theory and Practice," 3^rd Ed. Butterworth Heinemann, Oxford, UK; and in Horobin and

Kiernan, "Conn's biological stains: a handbook of dyes, stains and fluorochromes for us in biology and medicine," 10^th ed., Oxford: BIOS, ISBN 1859960995, 2002. The contents of the two bound references cited immediately above are incorporated by reference herein.

III. Counter Staining

Histochemical staining of a biological sample can include contacting the sample with a counterstain. In some embodiments, contacting the sample with a counterstain comprises contacting the sample with one or more of eosin Y, orange G, light green SF yellowish, Bismark Brown, fast green FCF, OA-6, EA25, EA36, EA50 and EA65. The formulas and methods of making such counterstains can be found, for example, in the StainsFile (an internet resource for histotechnologists maintained by Bryan Llewellyn); Kiernan, "Histological and Histochemical methods: Theory and Practice," 3^rd Ed. Butterworth Heinemann, Oxford, UK; and in Horobin and Kiernan, "Conn's biological stains: a handbook of dyes, stains and fluorochromes for us in biology and medicine," 10^th ed., Oxford: BIOS, ISBN 1859960995, 2002. In particular embodiments, the method is used to stain a tissue section or a cytology sample mounted on a microscope slide. In particular embodiments further including a counterstaining step, the method can be a hematoxylin and eosin (H&E) staining method or a PAP staining method, and more particularly an automated H&E or PAP staining method. IV. Gradient Staining

FIGS. 1-4 illustrate gradient staining results that occur when using certain protocols involving hematoxylin/hematein staining compositions. It currently is believed that gradient staining occurs when a staining species is produced during the staining process that is more reactive than is typically provided by a hematoxylin- hematein staining composition. For example, it may be that a polymerized hematein species is produced during the staining process. Production of the polymerized species may be induced by a pH change to the hematoxylin/hematein staining composition as a result of a processing step. Thus, certain embodiments of the present invention concern process step changes that substantially reduce or eliminate any pH changes that occur which produce the gradient staining result. Alternatively, wash compositions, such as compositions that are buffered to provide a pH at or about that of the hematoxylin/hematein staining composition, can be used to address the gradient staining results. And, combinations of process step changes and wash composition changes also can be used to address the gradient staining results.

A. Prior Automated Process Steps

A first embodiment of a method for reducing or substantially eliminating gradient hematoxylin staining for automated systems is to change a process step or steps that produce a pH gradient on a slide. Various aspects of automated systems are disclosed in U.S. Patent Nos. 5,654,200, 6,582,962, 6,855,552, 7,270,785, 7,303,725 and 7,378,055, each of which is incorporated herein by reference.

A first commercial process that has been used for an automated system, such as the Symphony^® platform provided by Ventana Medical Systems, Inc., is illustrated by the software flow chart of FIG. 6. With reference to FIG. 6, the first step is deparaffinization step 12. Deparaffinization can be accomplished using any suitable protocol. Solely by way of example, embodiments of a method for deparaffinizing are described in U.S. Patent No. 6,855,559, assigned to Ventana Medical Systems, Inc., and incorporated herein by reference. Briefly, a paraffin- embedded biological sample on a glass microscope slide is heated using a heating element. Heating the sample can be used to accomplish various goals, such as to melt the inert material, including paraffin and/or to drive off any water which may be between the paraffin section and the glass to allow the charge of the tissue to adhere to the glass. The inert material may be removed from the slide by a applying to the sample a fluid suitable to dissolve the inert material. Reagents can be used instead of or in addition to heating the embedded biological samples. Suitable reagents include, but are not limited to, de-ionized water, citrate buffer (pH 6.0-8.0), Tris-HCl buffer (pH 6-10), phosphate buffer (pH 6.0-8.0), SSC buffer, APK Wash™, acidic buffers or solutions (pH 1-6.9), basic buffers or solutions (pH 7.1- 14), mineral oil, Norpar, canola oil, and PAG oil. Each of these reagents also may contain ionic or non-ionic surfactants such as Triton X-100, Tween, Brij, saponin and sodium dodecylsulfate.

Following the deparaffinization step, a deparaffinization rinse step 14 is performed. This step usually is performed using a lower alkyl (10 carbon atoms or fewer) alcohol, such as methanol, ethanol and/or isopropanol. For disclosed embodiments, this rinse step typically involved applying 4 milliliters of the selected alcohol to each slide.

The sample is then prepared for nuclear staining in nuclear stain prep step 16. This is accomplished by applying deionized water, 1.2 milliliters for certain working embodiments, to the sample. A hematoxylin nuclear stain composition is then applied to the sample in step 18, followed by an incubation step 20. For the light stain composition indicated above, about 0.8 milliliter of the hematoxylin nuclear stain composition is applied to the sample. The duration of incubation step 20 is any period of time that allows effective staining results; however, for embodiments using the Symphony^® platform, the incubation step typically is about 1 minute.

Following incubation step 20, the sample is subjected to a nuclear stain rinse step 22. Tissue samples that are treated with a hematoxylin stain often are washed with deionized water, having a pH of about 5-7. Other wash compositions also can be used. For example, certain working embodiments are directed to tissue samples that are treated with a hematoxylin stain and washed with a wash composition comprising diluted Tween^® 20, such as an aqueous composition comprising about 1:20 Tween 20 and deionized water, and/or ProClin^® 300. Tween^® 20 (also referred to as polysorbate 20, polyoxyethylene (20) sorbitan monolaurate or PEG(20)sorbitan monolaurate) is a surfactant having a molecular formula of C58H₁₁₄O₂6, that is used as a detergent and emulsifier in a number of pharmacological applications. Tween 20, available from Sigma- Aldrich, is distinguished from the other members in the Tween^® range by the length of the polyoxyethylene chain and the fatty acid ester moiety. The active ingredients of ProClin^® 300, available from Sigma- Aldrich, total 3%, minimum, and include 5-chloro-2-methyl-4-isothiazolin-3-one (RH-651), plus 2-methyl-4-isothiazolin-3-one (RH-573). The typical ProClin^® 300 formulation is 5-chloro-2-methyl-4-isothiazolin-3-one 2.30%, 2-methyl-4-isothiazolin-3-one 0.70%, modified glycol 93-95% and an alkyl carboxylate, 2-3%. An exemplary wash composition is provided below.

Table 3 - Hematoxylin Wash Composition

Hematoxylin staining protocols typically also require using bluing compositions. Thus, FIG. 6 illustrates treating the sample with a bluing preparation in step 24, followed by a bluing step 26. Any suitable bluing solution can be used, including an ammonia-bluing reagent available from Richard Allen Scientific, Kalamazoo, Michigan. A particular bluing composition used by Ventana Medical Systems, Inc. for its automated platforms has the following composition.

Table 4 - Hematoxylin Bluing Composition

With reference to this composition, Tris is an abbreviation for tris(hydroxymethyl)aminomethane, having a molecular formula of (HOCH₂^CNH₂, which is available from Sigma- Aldrich. ProClin^® 950 is also available from Sigma- Aldrich. "950" indicates that the active ingredients, 5-chloro-2-methyl-4- isothiazolin-3-one (RH-651), and 2-methyl-4-isothiazolin-3-one (RH-573), total about 9.5%.

Once the bluing steps have been completed, a bluing rinse step 28 is performed to remove bluing composition. The bluing rinse typically involves rinsing with about 16-17 milliliters of deionized water. These steps typically are sufficient to complete the nuclear stain using the hematoxylin hematein staining composition.

A person of ordinary skill in the art will appreciate that additional steps can be performed, as indicated in FIG. 6. For example, the cytoplasmic stain can be performed. As a result, in a cytoplasmic stain prep step 30, cytoplasmic stain preparation composition is applied to the sample, followed by cytoplasmic stain step 32. A cytoplasmic stain is allowed to incubate on the sample for effective period of time in step 34, followed by the differentiation step 36. An additional incubation step 38 is performed followed by dehydration step 40 by application of an effective amount of an alcohol. A solvent exchange step 42 is then performed followed by application of a protective reagent to the sample in the process step 44.

These process steps have been by Ventana Medical Systems, Inc. previously for hematoxylin staining using, for example, the Symphony platform. These process steps can result in gradient staining, particularly when the staining preparation is the dark staining composition provided above.

B. Automated Process Step Modifications

FIG. 7 is a flow diagram 100 illustrating process steps for automated systems that have been used to address gradient staining that may occur with hematoxylin- hematein staining protocols. All process steps of FIG. 7 can be practiced using the reagents/compositions stated above for FIG. 6. Process 100 begins with a deparaffinization step 102 followed by deparaffinization rinse step 104. The sample is then ready for nuclear stain preparation 106 by the addition of a wash composition. A hematoxylin-hematein stain composition is then applied in step 108. In contrast to the prior process illustrated with reference to FIG. 6, bluing composition is then substantially immediately added to the sample in step 110. Step 112 is a mixing step. Any mixing mechanism that can effectively mix the compositions, and thereby substantially reduce or preclude pH gradients that may occur along the slide, may be used in this mixing step 112. Step 112 was not used in prior processes. In a working embodiment, mixing was achieved by applying an air puff from a dispensing nozzle, or nozzles, of the automated system that are positioned adjacent to each slide, such as above the slide. For example, again in a working embodiment, following addition of a bluing composition from about five to about 10 air puffs, typically about 8 air puffs, were applied to the top surface of each slide at different positions along the slide to effectively mix the hematoxylin-hematein stain composition and the bluing reagent. Each air pulse has a duration effective to mix the reagents, such as from about 20 to about 150 milliseconds, more typically from about 50 to about 100 milliseconds. The air pulse also is applied at a pressure effective to mix the reagents. This pressure may vary, depending on various factors such as distance of application from the slide surface, but typically is from about 5 to about 20 psi. For certain disclosed working embodiments the air pressure was applied at from about 14 to about 16 psi, and working embodiments most typically involved dispensing at least one air pulse from a nozzle having a diameter of about 0.022 inch at about 15 p.s.i.

Following the mixing step 112, a nuclear stain rinse is then applied in step 116. In step 118, an acid wash prep step is conducted followed by an optional acid wash step 120. If an optional acid wash step is used, once the acid wash step has been completed, the acid wash must be rinsed off in acid wash rinse step 122. A second bluing prep step 124 is then applied to the sample, followed by a bluing step 126. Following the bluing step, a bluing rinse is applied in step 128.

A person of ordinary skill in the art will appreciate that, as with the embodiment illustrated by FIG. 6, additional steps can be performed. For example, as illustrated by FIG. 7, a cytoplasmic stain can be performed. As a result, cytoplasmic stain preparation composition is applied to the sample in cytoplasmic stain preparation step 130. The sample is then subjected to an incubation step 132. Incubation step 132 continues for an effective period, such as a period of about 2 minutes. Incubation step 132 is then followed by cytoplasmic stain step 134. Cytoplasmic stain step 134 is followed by a differentiation step 136. An additional incubation step 138 is performed followed by dehydration step 140. Dehydration is accomplished by application of an effective amount, about 23 milliliters in a working embodiment, of a lower alkyl alcohol. A solvent exchange step 142 is then performed followed by application of a protective reagent to the sample in process step 144.

B. Inducing pH Change in Process Compositions, Such as Wash and Bluing Compositions

The automated process step changes indicated above were useful for addressing gradient hematoxylin staining results that occur when using automated staining platforms, such as the Symphony^® platform provided by Ventana Medical Systems, Inc. However, this approach, while successfully addressing the gradient staining problem, is not the only embodiment disclosed in the present application for ameliorating hematoxylin gradient staining. Instead, any mechanism whereby the gradient staining that occurs with certain hematoxylin staining protocols is reduced or eliminated is part of the present invention.

For example, but without being bound by a theory of operation, it currently is believed that the addition of reagents to the hematoxylin stain can induce a pH change in the staining composition, and that such pH change causes polymerization of species present in the staining composition. The polymerized species appears to provide a darker staining result than does the hematoxylin staining composition prior to this pH-induced composition change. It also appears that the formation of the more intense staining species is a quick reaction. Thus, staining darkness is greater in regions in which the more intense staining species is first formed, and a gradient staining pattern results as formation of the more intense staining species radiates outwardly from that region in which the pH-induced staining change first occurs. Because this reaction is quite fast, any rinse process that occurs and which induces a pH change also produces a gradient staining result that is lighter adjacent an application point of the wash reagent to a darker stained portion distal from the wash application point. Any process that either substantially prevents or completely precludes the pH change from occurring, or which makes the pH-induced change occur substantially uniformly in time across the surface of a particular slide, also will alleviate or preclude hematoxylin gradient staining results.

For example, tissue samples may be treated with hematoxylin stain process reagents or compositions, such as wash reagents or compositions, that are at or about the pH of the hematoxylin stain itself, e.g. from about 1 to about 6, more typically from about 2 to about 3, more typically from about 2.1 to about 2.8, and even more typically from about 2.2 to about 2.7. Furthermore, such compositions can be buffered to a pH that is at or about the pH of the hematoxylin stain.

Prior processes often washed stained samples with deionized water, having a pH of about 5-7. The addition of a substantial amount of deionized water to a hematoxylin stain at a lower pH therefore can substantially change the pH of the sample, resulting in gradient staining.

As a result, washing with an aqueous composition that is otherwise non- reactive with the sample or other compositions applied to the sample, and having a pH approximately equal to the hematoxylin stain can be used to address gradient staining. As stated above, tissue samples that are treated with a hematoxylin stain may be washed with a wash composition, such as a wash composition comprising surfactants, such as Tween 20, and/or antimicrobials, such as ProClin 300. Thus, in some embodiments where the hematoxylin has a pH of from about 2 to about 3, any wash composition that can be buffered to approximately the same pH can be used. One example of a suitably buffered wash composition is provided below. Table 5 - Buffered Hematoxylin Wash Composition

Any suitable buffering solution/composition can be used. Suitable buffers can be determined by reference to Remington's Science of Pharmacy, which provides a list of USP Standard Buffer Solutions. Solely by way of example, and without limitation, a partial list of such buffers includes acetate, maleate, phosphate, glycine, citrate, glycylglycine, malate, succinate, proprionate, pyridine, piperazine, and formate. One working embodiment used 100 mM chloroacetate buffer at a pH of about 3.0.

In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the invention and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

Claims

We claim:

1. A method for staining a tissue sample for microscopic examination, comprising substantially reducing or preventing gradient hematoxylin staining of the tissue sample.

2. The method according to claim 1 comprising an automated staining method.

3. The method according to claim 1 comprising a manual staining method.

4. The method according to claim 2, comprising: dispensing a hematoxylin stain onto a tissue sample; dispensing a bluing composition onto the tissue sample to form a combined hematoxylin stain-bluing composition; and agitating the hematoxylin stain-bluing composition.

5. The method according to claim 4 wherein the hematoxylin stain comprises deionized water, ethylene glycol, hematoxylin dye, sodium iodate, aluminum sulfate hydrate and glacial acetic acid.

6. The method according to claim 4 wherein the hematoxylin stain comprises deionized water, ethylene glycol, hematoxylin dye, sodium iodate, aluminum sulfate, hydroquinone and β-cyclodextrin hydrate.

7. The method according to claim 4 wherein agitating comprises air agitation.

8. The method according to claim 7 wherein air agitation comprises dispensing plural air pulses onto a slide.

9. The method according to claim 1, comprising: dispensing a hematoxylin stain onto a tissue sample; incubating the tissue sample for a period of time effective to stain the tissue sample; and washing the sample with a wash solution having a pH at or about that of the hematoxylin stain.

10. The method according to claim 9 wherein the wash solution is an aqueous buffered solution having a pH of from about 2 to about 3.

11. The method according to claim 1 comprising contacting the tissue sample at least once with an aqueous solution comprising 0.1 to 2% hematoxylin for a period of time effective to stain the sample, and then washing the sample with a wash solution buffered to a pH of about 2 to about 3.

12. The method according to claim 2 wherein the automated system is substantially under computer control.

13. The method according to claim 12 where the automated system performs the following steps: deparaffinizing the sample, if required; staining the sample using a hematoxylin stain; adding a bluing reagent; and air mixing the hematoxylin stain and the bluing reagent.

14. The method according to claim 13 further comprising an acid wash and an acid wash rinse.

15. The method according to claim 13 where the automated system further performs at least one additional staining protocol.

16. The method according to claim 15 where the at least one additional staining protocol is cytoplasmic staining.

17. An automated method for staining a tissue sample for microscopic examination that substantially reduces or prevents gradient hematoxylin staining, comprising: adding at least one slide having a sample to an automated staining system; deparaffinizing the sample, if required; applying a hematoxylin stain composition to the sample; applying a bluing reagent or composition to the sample; and air mixing the hematoxylin stain and the bluing reagent on the slide.

18. The method according to claim 17 where air mixing comprises dispensing at least one air pulse onto the sample.

19. The method according to claim 18 comprising dispensing plural air pulses onto the slide.

20. The method according to claim 18 where the at least one air pulse has a duration of from about 50 to about 100 milliseconds.

21. The method according to claim 18 comprising dispensing at least one air pulse at from about 14 to about 16 psi.

22. The method according to claim 21 comprising dispensing at least one air pulse at about 15 psi.

23. The method according to claim 17 wherein the automated staining system is substantially under computer control.

24. The method according to claim 17 wherein the automated system further performs at least one additional staining protocol.

25. The method according to claim 17 wherein the at least one additional staining protocol is cytoplasmic staining.

26. An automated method for staining a tissue sample for microscopic examination that substantially reduces or prevents gradient hematoxylin staining, comprising: adding at least one slide having a sample to an automated staining system that is substantially under computer control; deparaffinizing the sample, if required; applying a hematoxylin stain composition to the sample; applying a bluing reagent or composition to the sample; air mixing the hematoxylin stain and the bluing reagent on the slide by dispensing plural air pulses onto the slide, each air pulse having a duration of from about 50 to about 100 milliseconds, and being applied at from about 14 to about 16 psi; and performing at least one additional staining protocol on the sample.

27. The method according to claim 26 wherein the at least one additional staining protocol is cytoplasmic staining.

28. An automated method for staining a tissue sample for microscopic examination, the automated method comprising one or more tangible computer- readable media wherein said one or more tangible computer-readable media comprises stored computer-executable instructions for causing a computer to perform a method, the method comprising: dispensing a hematoxylin stain onto a tissue sample; dispensing a bluing composition onto the tissue sample to form a combined hematoxylin stain-bluing composition; and agitating the hematoxylin stain-bluing composition thereby providing an automated method for staining a tissue sample for microscopic examination.

29. An automated method for staining a tissue sample for microscopic examination, the automated method comprising one or more tangible computer- readable media wherein the one or more tangible computer-readable media comprises stored computer-executable instructions for causing a computer to perform a method, the method comprising: deparaffinizing the sample, if required; applying a hematoxylin stain composition to the sample; applying a bluing reagent or composition to the sample; air mixing the hematoxylin stain and the bluing reagent on the slide by dispensing plural air pulses onto the slide, each air pulse having a duration of from about 50 to about 100 milliseconds, and being applied at from about 14 to about 16 psi; and performing at least one additional staining protocol on the sample thereby providing an automated method for staining a tissue sample for microscopic examination.

30. A hematoxylin stain wash composition having a pH of from about 2 to about 3.