Semi-synthesis and use of racemic hematoxylin

Abstract

To provide a hematoxylin formulation comprising racemic hematoxylin.SOLUTION: A first aspect of the present disclosure is a hematoxylin formulation including racemic hematoxylin and at least one additive (e.g., at least one additive selected from a solvent, a chemical oxidant, a mordant, a stabilizer, and an antioxidant). Another aspect is a method for staining a biological sample, comprising contacting the biological sample with a racemic hematoxylin formulation. In some embodiments, the racemic hematoxylin formulation includes a solvent, hematoxylin, an amount of a chemical oxidant sufficient to convert at least a portion of the hematoxylin into hematein, a mordant, an antioxidant, and a stabilizer. In some embodiments, the stabilizer is selected from the group consisting of a polysaccharide, a cryptand, a cryptophane, a cavitand, a crown ether, a dendrimer, a nanotube, a calixarene, a valinomycin, and a nigericin.SELECTED DRAWING: None

Description

[Background technology]

[0001] Hematoxylin has been described as the most important and most widely used dye in histology, histochemistry, histopathology, and cytology. Several histochemical staining protocols, including hematoxylin and eosin (H&E) and Papanicolaou (PAP) stains, rely on the dye hematoxylin to stain cytological and tissue samples. In particular, hematoxylin staining of cell nuclei is used by pathologists to detect the presence of malignant and / or metastatic cells in tumor biopsy samples.

[0002] Hematoxylin is a naturally occurring compound found in the red heartwood of the Haematoxylum tree. Hematoxylin itself is colorless in aqueous solution and is not the active component that stains tissue components. Rather, hematein, the oxidation product of hematoxylin, is the active staining component of hematoxylin solutions, especially when complexed with a mordant. For example, to produce a functional dye, hematoxylin can be oxidized to hematein, followed by aluminum (Al +3 ), iron (Fe +3 ), and chromium (Cr +3 Hematein is bound to one of several metal ions, including thiamin (H2O), thiamin (H2O), and thiamin (H2O). Hematein is naturally produced by exposure to air and sunlight. The natural process is called "maturation" and can take three months or more to obtain a solution suitable for staining cells.

[0003] Hematoxylin staining can be performed manually using a dip-and-dunk technique or using an automated system such as the Symphony® automated system from Ventana Medical Systems, Inc. The staining process generally involves (a) removing paraffin from a specimen fixed on a microscope slide and hydrating the specimen by immersion in water; (b) applying hematoxylin in a specific formulation to stain cell nuclei; (c) removing excess hematoxylin by rinsing with water; (d) contacting the slide with a concentrated solution with a pH value greater than 5.0 to turn the hematoxylin blue (e.g., a bluing solution); and (e) removing the bluing solution by rinsing with water. Summary of the Invention

[0004] The present disclosure provides hematoxylin preparations comprising racemic hematoxylin. In some embodiments, the racemic hematoxylin preparations are suitable for staining the nuclei of cells in biological samples. In some embodiments, the racemic hematoxylin preparations are suitable for staining rough endoplasmic reticulum, ribosomes, collagen, myelin, elastic fibers, and acidic mucins. In some embodiments, the racemic hematoxylin preparations are suitable as a counterstain for CISH (chromogenic in situ hybridization) or for visualizing morphology around specific antigen-antibody complexes (chromogenic immunohistochemistry). Applicant unexpectedly discovered that staining with racemic hematoxylin is functionally equivalent to staining with commercially available enantiopure hematoxylin.

[0005] A first aspect of the present disclosure is a hematoxylin formulation comprising racemic hematoxylin and at least one additive (e.g., at least one additive selected from a solvent, a chemical oxidizing agent, a mordant, a stabilizer, and an antioxidant). In some embodiments, the hematoxylin formulation comprises at least two additives. In some embodiments, the hematoxylin formulation comprises at least three additives. In some embodiments, the hematoxylin formulation comprises racemic hematoxylin, a solvent, a chemical oxidizing agent, a mordant, a stabilizer, and an antioxidant. In some embodiments, a certain amount of the chemical oxidizing agent present in the hematoxylin formulation is sufficient to convert at least a portion of the racemic hematoxylin to hematein. In some embodiments, the solvent is an aqueous solvent. In some embodiments, the antioxidant is a water-soluble antioxidant. In some embodiments, the water-soluble antioxidant comprises hydroquinone.

[0006] In some embodiments, the solvent comprises one or more of water, a lower alkanol, and a polyol. In some embodiments, the solvent comprises water and a polyol. In some embodiments, the polyol is selected from the group consisting of propylene glycol, poly(ethylene glycol), and poly(propylene glycol).

[0007] In some embodiments, between 1% and 50% of the racemic hematoxylin in the hematoxylin formulation is oxidized to hematein by a chemical oxidizing agent. In some embodiments, the chemical oxidizing agent includes one or more of sodium iodate, mercury oxide, potassium permanganate, potassium periodate, and hydrogen peroxide. In some embodiments, the chemical oxidizing agent includes sodium iodate. In some embodiments, the molar ratio of hematoxylin to oxidizing agent in the composition ranges from about 6:1 to about 1:1. In some embodiments, the mordant includes 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 includes aluminum sulfate.

[0008] In some embodiments, the stabilizer comprises a cyclodextrin or a cyclodextrin derivative, hi some embodiments, the cyclodextrin or cyclodextrin derivative is one or more of a β-cyclodextrin and a β-cyclodextrin derivative.

[0009] In some embodiments, the hematoxylin formulation further comprises an acid. In some embodiments, no acid is added to the hematoxylin formulation.

[0010] Another aspect of the present disclosure is a method for staining a biological sample, comprising contacting the biological sample with a racemic hematoxylin preparation. In some embodiments, the racemic hematoxylin preparation comprises a solvent, hematoxylin, a chemical oxidizing agent in an amount sufficient to convert at least a portion of the hematoxylin to hematein, a mordant, an antioxidant, and a stabilizer. In some embodiments, the stabilizer is selected from the group consisting of polysaccharides, cryptands, cryptophanes, cavitands, crown ethers, dendrimers, nanotubes, calixarenes, valinomycin, and nigericin.

[0011] In some embodiments, the method further comprises contacting the sample with a counterstain. In some embodiments, the counterstain is selected from the group consisting of Eosin Y, Orange G, Light Green SF Yellow, Bismarck Brown, and Fast Green FCF. In some embodiments, contacting the sample with the racemic hematoxylin preparation comprises a progressive hematoxylin staining protocol. In some embodiments, contacting the sample with the racemic hematoxylin preparation comprises a regressive hematoxylin staining protocol. In some embodiments, the method is automated. In some embodiments, the biological sample is supported on a substrate. In some embodiments, the substrate comprises a microscope slide.

[0012] In some embodiments, the stabilizer has an aqueous solubility of greater than about 100 mg / mL at about 25° C. In some embodiments, the antioxidant is selected from the group consisting of hydroquinone, n-alkyl gallates, reducing sugars, benzoates, hydroxybenzoates, sulfites, metabisulfites, citric acid, tartaric acid, lactic acid, erythorbic acid, ascorbic acid, uric acid, tannic acid, one or more chelating agents, coral hydrate, derivatives and salts thereof.

[0013] Another aspect of the present disclosure is a pH-reduced hematoxylin formulation comprising racemic hematoxylin and an acid in an amount ranging from about 0.2% to about 4% of the total volume of the pH-reduced hematoxylin formulation, wherein the pH-reduced hematoxylin formulation has a pH value of less than about 2.4. In some embodiments, the acid is selected from the group consisting of hydrochloric acid, sulfuric acid, perchloric acid, and nitric acid. In some embodiments, the pH value is less than about 2.3. In some embodiments, the pH value is less than about 2.2. In some embodiments, the pH value is less than about 2.1. In some embodiments, the pH value is in the range of about 2.1 to about 2.2.

[0014] In some embodiments, the reduced pH hematoxylin formulation further comprises a mordant and an oxidizing agent. In some embodiments, the reduced pH hematoxylin formulation further comprises a stabilizer. In some embodiments, the stabilizer comprises a polyol. In some embodiments, the polyol is selected from the group consisting of propylene glycol, ethylene glycol, and mixtures thereof.

[0015] In some embodiments, the formulation consists essentially of racemic hematoxylin, a mordant, an oxidizing agent, and an acid. In some embodiments, the oxidizing agent comprises sodium iodate. In some embodiments, the mordant comprises aluminum.

[0016] Another aspect of the present disclosure is a method of staining a biological sample, comprising increasing the pH value of an aliquot of a pH-reduced hematoxylin formulation to provide a racemic hematoxylin staining solution having a pH value in the range of about 2.4 to about 2.6, and contacting the biological sample with the hematoxylin staining solution having a pH value in the range of about 2.4 to about 2.6. In some embodiments, the pH value of the aliquot of the pH-reduced hematoxylin formulation is increased by adding a readjustment solution comprising a strong base or buffer.

[0017] In some embodiments, the strong base is selected from the group consisting of sodium hydroxide, potassium hydroxide, lithium hydroxide, and ammonia.

[0018] In some embodiments, the reconditioning solution further comprises an additive selected from the group consisting of a polyol, an oxidizing agent, a mordant, and any combination thereof. In some embodiments, the pH value of the pH-reduced hematoxylin formulation is increased prior to contacting the biological sample with the hematoxylin staining solution. In some embodiments, the biological sample comprises a histological sample or a cytological sample.

[0019] In another aspect of the present disclosure, a kit is provided that includes a first component and a second component. In some embodiments, the first component includes a pH-reduced hematoxylin formulation that includes racemic hematoxylin and an acid in an amount ranging from about 0.1% to about 10% of the total volume of the pH-reduced hematoxylin formulation, where the pH-reduced hematoxylin formulation has a pH value of less than about 2.4. The second component includes a strong base or buffer and is provided in an amount relative to the first component such that when the first and second components are combined, the pH value of the pH-reduced hematoxylin formulation is raised to above about 2.4.

[0020] In some embodiments, the second component is provided in an amount relative to the first component such that, when the first and second components are mixed, the pH value of the pH-reduced hematoxylin formulation is raised to above about 2.5. In some embodiments, the second component is provided in an amount relative to the first component such that, when the first and second components are mixed, the pH value of the pH-reduced hematoxylin formulation is in the range of about 2.45 to about 2.54. In some embodiments, the second component is provided in an amount relative to the first component such that, when the first and second components are mixed, the pH value of the pH-reduced hematoxylin formulation is in the range of about 2.5 to about 2.6.

[0021] In some embodiments, the first component further comprises a mordant and an oxidizing agent. In some embodiments, the first component further comprises a shelf-life extender. In some embodiments, the shelf-life extender is a polyol. In some embodiments, the polyol is selected from the group consisting of propylene glycol, ethylene glycol, and mixtures thereof.

[0022] In some embodiments, the reduced pH hematoxylin formulation consists essentially of hematoxylin, a mordant, an oxidizing agent, and an acid. In some embodiments, the second component comprises a strong base selected from the group consisting of sodium hydroxide, potassium hydroxide, and lithium hydroxide. In some embodiments, the second component comprises a buffer selected from the group consisting of cyanoacetate, fumarate, and sulfate.

[0023] In some embodiments, the kit further comprises a third component comprising a counterstain, hi some embodiments, the counterstain is selected from the group consisting of Eosin Y, Orange G, Light Green SF Yellow, Bismarck Brown, and Fast Green FCF.

[0024] In another aspect of the present disclosure, a system for staining a biological sample mounted on a substrate is provided, comprising: a first container containing a pH-reduced hematoxylin formulation (racemic hematoxylin) and an acid in an amount ranging from about 0.1% to about 10% of the total volume of the pH-reduced hematoxylin formulation, wherein the pH-reduced hematoxylin formulation has a pH value less than about 2.4; and a second container containing a readjustment solution. In some embodiments, the first and second containers are fluidly connected to a mixing container such that the pH-reduced hematoxylin formulation and the readjustment solution can be mixed to provide a hematoxylin staining solution. In some embodiments, the system further includes a substrate holder for holding the biological sample mounted on the substrate, the substrate holder being in fluid communication with the mixing container such that the hematoxylin staining solution can be applied to the biological sample.

[0025] For a general understanding of the features of the present disclosure, reference is made to the drawings, wherein like reference numerals are used throughout to identify identical elements. [Brief explanation of the drawings]

[0026] [Figure 1A] FIG. 1 shows an autoscaled HPLC chromatogram of a solution containing commercially available hematoxylin, and the resulting peaks. [Figure 1B] Zoomed-in view of the peak results of the HPLC chromatogram of FIG. 1A for each component of the solution. [Figure 2A] FIG. 1 shows an autoscaled HPLC chromatogram of a solution containing racemic hematoxylin and peak results. [Figure 2B] Zoomed-in view of the peak results of the HPLC chromatogram of Figure 2A for each component of the solution. [Figure 3A] Figure showing tissue samples stained with commercial hematoxylin. [Figure 3B] Figure showing tissue samples stained with commercial hematoxylin. [Figure 4A] Figure showing tissue samples stained with racemic hematoxylin. [Figure 4B] Figure showing tissue samples stained with racemic hematoxylin. [Figure 5A] Comparison of 20x commercial hematoxylin with 20x synthetic hematoxylin (Trichrome Hematoxylin) in kidney glomerular tissue. Staining with synthetic hematoxylin provides clearer images of the details and overlap of larger cell nuclei. [Figure 5B] Comparison of 20x commercial hematoxylin with 20x synthetic hematoxylin (Trichrome Hematoxylin) in kidney glomerular tissue. Staining with synthetic hematoxylin provides clearer images of the details and overlap of larger cell nuclei. [Figure 6A] Comparison of 20x commercial hematoxylin with 20x synthetic hematoxylin (mucicarmine-iron-hematoxylin) in colon tissue. Staining with synthetic hematoxylin provides clearer images of the details and overlap of larger cell nuclei. [Figure 6B] Comparison of 20x commercial hematoxylin with 20x synthetic hematoxylin (mucicarmine-iron-hematoxylin) in colon tissue. Staining with synthetic hematoxylin provides clearer images of the details and overlap of larger cell nuclei. [Figure 7A] Comparison of 20x commercial hematoxylin with 20x synthetic hematoxylin (hematoxylin II) in colon tissue. Staining with synthetic hematoxylin provides clearer images of the details and overlap of larger cell nuclei. [Figure 7B] Comparison of 20x commercial hematoxylin with 20x synthetic hematoxylin (hematoxylin II) in colon tissue. Staining with synthetic hematoxylin provides clearer images of the details and overlap of larger cell nuclei. [Figure 8A] Comparison of 20x commercial hematoxylin with 20x synthetic hematoxylin (hematoxylin II) in kidney glomerular tissue. Staining with synthetic hematoxylin provides clearer images of the details and overlap of larger cell nuclei. [Figure 8B]Comparison of 20x commercial hematoxylin with 20x synthetic hematoxylin (hematoxylin II) in kidney glomerular tissue. Staining with synthetic hematoxylin provides clearer images of the details and overlap of larger cell nuclei. [Figure 9A] Comparison of 20x commercial hematoxylin with 20x synthetic hematoxylin (hematoxylin II) in tonsil tissue. Staining with synthetic hematoxylin provides a clearer image of the details and overlap of larger cell nuclei. [Figure 9B] Comparison of 20x commercial hematoxylin with 20x synthetic hematoxylin (hematoxylin II) in tonsil tissue. Staining with synthetic hematoxylin provides a clearer image of the details and overlap of larger cell nuclei. [Figure 10A] Comparison of 20x commercial hematoxylin with 20x synthetic hematoxylin (hematoxylin-eosin) in kidney glomerular tissue. Staining with synthetic hematoxylin provides clearer images of the details and overlap of larger cell nuclei. [Figure 10B] Comparison of 20x commercial hematoxylin with 20x synthetic hematoxylin (hematoxylin-eosin) in kidney glomerular tissue. Staining with synthetic hematoxylin provides clearer images of the details and overlap of larger cell nuclei. [Figure 11A] Comparison of 20x commercial hematoxylin with 20x synthetic hematoxylin (hematoxylin-eosin) in muscle-vascular tissue. Staining with synthetic hematoxylin provides clearer images of the details and overlap of larger cell nuclei. [Figure 11B] Comparison of 20x commercial hematoxylin with 20x synthetic hematoxylin (hematoxylin-eosin) in muscle-vascular tissue. Staining with synthetic hematoxylin provides clearer images of the details and overlap of larger cell nuclei. [Figure 12A]Comparison of 20x commercial hematoxylin with 20x synthetic hematoxylin (hematoxylin-eosin) in colon tissue. Staining with synthetic hematoxylin provides clearer images of the details and overlap of larger cell nuclei. [Figure 12B] Comparison of 20x commercial hematoxylin with 20x synthetic hematoxylin (hematoxylin-eosin) in colon tissue. Staining with synthetic hematoxylin provides clearer images of the details and overlap of larger cell nuclei. [Figure 13A] Comparison of 20x commercial hematoxylin with 20x synthetic hematoxylin (hematoxylin-eosin) in sebaceous gland and hair follicle tissue. Staining with synthetic hematoxylin provides clearer images of the details and overlap of larger cell nuclei. [Figure 13B] Comparison of 20x commercial hematoxylin with 20x synthetic hematoxylin (hematoxylin-eosin) in sebaceous gland and hair follicle tissue. Staining with synthetic hematoxylin provides clearer images of the details and overlap of larger cell nuclei. [Figure 14A] Comparison of 20x commercial hematoxylin with 20x synthetic hematoxylin (hematoxylin-eosin) in skin tissue. Staining with synthetic hematoxylin provides clearer images of the details and overlap of larger cell nuclei. [Figure 14B] Comparison of 20x commercial hematoxylin with 20x synthetic hematoxylin (hematoxylin-eosin) in skin tissue. Staining with synthetic hematoxylin provides clearer images of the details and overlap of larger cell nuclei. [Figure 15A] Comparison of 20x commercial hematoxylin with 20x synthetic hematoxylin (hematoxylin-eosin) in tonsil tissue. Staining with synthetic hematoxylin provides clearer images of the details and overlap of larger cell nuclei. [Figure 15B]Comparison of 20x commercial hematoxylin with 20x synthetic hematoxylin (hematoxylin-eosin) in tonsil tissue. Staining with synthetic hematoxylin provides clearer images of the details and overlap of larger cell nuclei. DETAILED DESCRIPTION OF THE INVENTION

[0027] It should also be understood that, unless expressly indicated to the contrary, in any method claimed herein that includes multiple steps or actions, the order of the method steps or actions is not necessarily limited to the order in which the method steps or actions are recited.

[0028] References herein to "one embodiment," "an embodiment," "an exemplary embodiment," etc., may indicate that the described embodiment may include a particular feature, structure, or characteristic, but all embodiments may or may not include that particular feature, structure, or characteristic. Moreover, such phrases do not necessarily refer to the same embodiment. Furthermore, when a particular feature, structure, or characteristic is described in connection with one embodiment, it is believed to be within the knowledge of one of ordinary skill in the art to affect such feature, structure, or characteristic in connection with other embodiments, whether or not explicitly stated.

[0029] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly dictates otherwise. The term "comprising" is defined inclusively, such that "including A or B" means including A, B, or A and B.

[0030] As used herein and in the claims, "or" should be understood to have the same meaning as "and / or," as defined above. For example, when separating items in a list, "or" or "and / or" shall be construed as being inclusive (e.g., including at least one) and also as including more than one of a number of elements or lists of elements, and, optionally, additional unlisted items. Only terms clearly indicated otherwise, such as "only one of" or "exactly one of," or, when used in the claims, "consisting of," refer to the inclusion of exactly one element of a number of elements or lists of elements. Generally, as used herein, the term "or" shall only be construed as indicating exclusive alternatives (e.g., "either," "one of," "only one of," or "exactly one of,") when preceded by exclusive terms, such as "either," "one of," "only one of," or "exactly one of." When used in the claims, "consisting essentially of" shall have its ordinary meaning as used in the field of patent law.

[0031] Terms such as "comprising," "including," and "having" are used interchangeably and have the same meaning. Similarly, terms such as "comprising," "including," and "having" are used interchangeably and have the same meaning. Specifically, each term is defined consistent with the general U.S. patent law definition of "comprising / including" and, therefore, is to be interpreted as being open terminology meaning "at least the following" and not excluding additional features, limitations, aspects, etc. Thus, for example, "a device having components a, b, and c" means that the device includes at least components a, b, and c. Similarly, the phrase "a method comprising steps a, b, and c" means that the method includes at least steps a, b, and c. Furthermore, although steps and processes may be outlined in a particular order herein, one of ordinary skill in the art will recognize that the order of steps and processes may vary.

[0032] As used herein and in the claims, the phrase "at least one" in connection with a list of one or more elements should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of every element specifically listed in the list of elements, and not excluding any combination of elements in the list of elements. This definition also allows for elements other than those specifically identified in the list of elements to which the phrase "at least one" refers, can optionally be present, whether related to the specifically identified elements or not. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B" or, equivalently, "at least one of A and / or B") refers, in one embodiment, to at least one, optionally more than one A, and no B (and optionally including elements other than B); in another embodiment, to at least one, optionally more than one B, and no A (and optionally including elements other than A); in yet another embodiment, to at least one, optionally more than one A, and at least one, optionally more than one B (and optionally including other elements); and so forth.

[0033] The term "antioxidant" refers to an atom or molecule that has a greater redox 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 may have a greater oxidizing power than hematein, thus helping to prevent the oxidation of hematein to oxyhematein. Furthermore, an antioxidant can also function as a reducing agent, e.g., converting oxyhematein back to hematein. The antioxidant can be present in the disclosed formulations at a concentration ranging from about 1 mM to about 1 M, e.g., from about 5 mM to about 500 mM, e.g., from about 50 mM to about 150 mM.

[0034] The term "aqueous solvent" refers to a composition that has water as its primary component and is liquid at room temperature. A mixture of water and one or more lower alkanols or polyols having a water content of about 50% or more by volume is an example of an aqueous solvent.

[0035] The term "biological sample" refers to any sample obtained or otherwise derived from a biological entity such as an animal, e.g., a sample obtained from a human or a veterinary animal such as a dog, cat, horse, or cow. Examples of biological samples include cytological samples, tissue samples, and biological fluids. Non-limiting examples of biological samples include blood, urine, pre-ejaculate fluid, nipple aspirate, semen, milk, sputum, mucus, pleural fluid, pelvic fluid, synovial fluid, ascites fluid, body cavity washings, eye brushings, skin scrapings, buccal swabs, vaginal swabs, Pap smears, rectal swabs, aspirates, needle biopsies, sections of tissue obtained, e.g., at surgery or autopsy, plasma, serum, cerebrospinal fluid, lymph, sweat, tears, saliva, tumors, organs, and samples obtained from in vitro cell or tissue culture. Typically, the sample is a biopsy sample that has been fixed, processed to remove water, and embedded in paraffin or another suitable waxy substance for sectioning into tissue sections. The biological sample can be mounted on a substrate, such as a microscope slide, for treatment and / or examination.

[0036] The term "mordant" refers to an ionic metal species capable of forming a complex (e.g., a cationic complex) that serves to bind a dye (e.g., hematein) to specific cellular components such as nuclear DNA, myelin, elastic and collagen fibers, muscle striations, and mitochondria. Examples of mordants include aluminum (e.g., in the form of alum, such as aluminum sulfate, potassium aluminum sulfate, or ammonium aluminum sulfate), iron, tungsten, zirconium, bismuth, molybdenum (phosphomolybdate or molybdate), and vanadium (vanadate).

[0037] The term "oxidizing agent" refers to an atom or molecule having a greater redox potential than a second molecule, e.g., a redox potential greater than that of hematoxylin, such that it reacts with hematoxylin to oxidize it to hematein. Oxidizing agents include naturally occurring molecular oxygen in the atmosphere, which diffuses into and oxidizes hematoxylin, and "chemical oxidizing agents" that actively combine with hematoxylin (usually in solution) to convert at least a portion of the hematoxylin to hematein. Examples of useful chemical oxidizing agents include one or more of iodates (e.g., sodium iodate and potassium iodate), mercury oxide, permanganates (e.g., potassium permanganate), periodates (e.g., sodium periodate and potassium periodate), and peroxides (e.g., hydrogen peroxide). In certain embodiments, the chemical oxidizing agent comprises sodium iodate.

[0038] The term "stabilizer" refers to an organic or inorganic molecule, composite, or material having an internal cavity or groove portion, more specifically, a molecule having an internal cavity or groove portion capable of accommodating at least a portion of a hematein or other dye molecule.

[0039] Overview The present disclosure provides hematoxylin preparations containing racemic hematoxylin. In some embodiments, the racemic hematoxylin preparations are suitable for staining the nuclei of cells in biological samples. Applicant unexpectedly discovered that staining with racemic hematoxylin is functionally equivalent to staining with commercially available enantiopure hematoxylin. This result is surprising, as chirality is paramount to almost all chemical processes in biological systems. It has also been shown to be inherently present in the structure of polynucleotides such as DNA and RNA, both at the molecular and supramolecular scales, and constitutes a primary target for hematoxylin-based anionic stains.[1,2] Their asymmetric D-ribose and D-2-deoxyribose units contain several stereocenters and sites with chiral environments suitable for hydrogen bonding, electrostatic interactions, or coordinate binding of chemical agents.[12e] Furthermore, their helical nature imparts another level of chirality to tertiary structures, as can be observed, for example, in B-DNA.

[0040] Chirality plays an important role in the function of DNA and RNA, guiding their interactions with other chiral and achiral chemical agents, such as enzymes or small molecules like dyes.[1,12e] Therefore, the interactions between a vast number of drugs, dyes, and organometallic complexes and different DNA conformations have been the subject of numerous studies.[2,3,4,5] Enantioselectivity of DNA binding is a crucial parameter in processes such as staining with chiral dyes[2,5,6] or treating diseases with enantiopure drugs[4], demonstrating the ability to control even the occurrence of undesirable side effects such as toxicity and mutagenicity. It is well known that enantiomers of natural products bind and react with DNA in different ways.

[0041] In this context, the enantiomers of duocarmycin A, a natural product isolated from Streptomyces species that alkylates DNA with high potency, represent a prominent example. Boger et al. demonstrated that (+)-duocarmycin A binds more selectively and alkylates DNA 110-fold more efficiently than (-)-duocarmycin A.[9] Daunorubicin, another prominent natural product and highly active anticancer drug, intercalates into DNA with its daunomycin aglycone unit and binds to the minor groove of B-DNA using its D-daunosamine residue.

[10] A recent study found that (+)-daunorubicin has a 21-fold increased binding affinity for specific sequences, such as poly[(dGdC)]2, in B-DNA compared to its (-)-enantiomer.

[11] Additionally, a wide variety of monomeric and linked polyamides have been used to elucidate the effect of chirality on their ability to bind to polynucleotide structures.

[12] In this group of molecules, the binding affinity of the R-enantiomer to B-DNA is generally strongly enhanced.

[0042] Chiral recognition of DNA by enantiopure dyes has been most intensively studied for helicene and other substituted planar polycyclic hydrocarbons, such as pyrene and anthracene derivatives.[3,6] Thus, the P-enantiomer of helicene, modified with a protonated amino group, exhibits discrimination in binding between B-DNA and Z-DNA.[5] A series of chiral cations[4] have been demonstrated in helicenes with binding affinities for dsDNA factorization ranging from 1.2 to 2.3.[3] Due to the general importance of DNA-metal interactions, chiral inorganic and organometallic complexes have also been extensively investigated for their binding properties to DNA-like structures. For example, certain Pt complexes of 1,2-diaminocyclohexane are significantly less toxic and more effective anticancer drugs, utilizing the R,R isomer rather than the S,S isomer.[7] Similarly, enantioselectivity in binding has been found for numerous other chiral metal complexes with iron, rhodium, ruthenium, and osmium centers, and they have been used as tools to probe various DNA conformations. [8] As a prime example of a hematoxylin-based staining formulation, hemalum itself is an organometallic complex of the Lewis acid Al3+ with hematein, the latter still possessing one stereocenter, making hemalum chiral in all of its different pH-dependent compositions and ligand spheres.[1]

[0043] Recent research by J.A. Kiernan suggests that red hemalum cations are electrostatically attracted to DNA phosphate anions, resulting in the in situ precipitation of blue compounds when stained preparations turn blue at pH values above 5.5; however, the nature of the hemalum-DNA interaction before and after bluing has not been fully elucidated. [2] Given the above chemical principles and scientific findings, a skilled chemist would assume that the use of pure hematoxylin enantiomers in histological staining would result in different staining intensities due to their different binding affinities to DNA. Based on this, the use of racemic hematoxylin in histology rather than the pure natural enantiomer should result in weaker or stronger staining, depending on which isomer forms the Al3+ complex with greater DNA affinity. Because histological analysis in tissue diagnosis remains a matter of subjective visual judgment, staining properties different from those of preparations made with pure (+)-hematoxylin would call into question the histologist's individual operating experience and the accuracy of their reported results. Only the nearly identical behavior of (+)-hematoxylin and (-)-hematoxylin on DNA would make the racemate worthwhile for use in hematoxylin-based tissue diagnostics.

[0044] Racemic hematoxylin preparations The present disclosure provides hematoxylin formulations, including racemic hematoxylin (hereinafter "racemic hematoxylin formulations"). Generally, racemic hematoxylin formulations include racemic hematoxylin and at least one additional component. In some embodiments, the additional component is selected from a stabilizer, a solvent, an antioxidant, an oxidizing agent, a mordant, and an acid. In some embodiments, racemic hematoxylin formulations include racemic hematoxylin and at least two additional components. In some embodiments, racemic hematoxylin formulations include racemic hematoxylin and at least three additional components. In some embodiments, racemic hematoxylin formulations include racemic hematoxylin and at least four additional components.

[0045] For example, in some embodiments, the racemic hematoxylin formulation includes racemic hematoxylin, a solvent, a chemical oxidizing agent, and a mordant. As another example, in some embodiments, the racemic hematoxylin formulation includes racemic hematoxylin, a solvent, and an acid. As yet another example, in some embodiments, the racemic hematoxylin formulation includes racemic hematoxylin, a solvent, a chemical oxidizing agent, a mordant, and one or both of a stabilizer and an antioxidant. As yet another example, in some embodiments, the racemic hematoxylin formulation includes racemic hematoxylin, a solvent, a chemical oxidizing agent, a mordant, a stabilizer, and an antioxidant. As yet another example, in some embodiments, the racemic hematoxylin formulation includes two or more different antioxidants, for example, two or more water-soluble antioxidants. As yet another example, in some embodiments, the racemic hematoxylin formulation includes one or more stabilizers and one or more antioxidants.

[0046] Additional ingredients suitable for use within any of the disclosed racemic hematoxylin formulations are described in Avwioro et al. "Histochemical Uses Of Hematoxylin - A Review," JPCS Vol (1), April-June 2011, and Bryan D. Llewellyn, "Hematoxylin Formulae," http: / / stainsfile.info, October 2013, the entire disclosures of which are hereby incorporated by reference.

[0047] Racemic Hematoxylin Hematoxylin has the structure set forth below: Racemic hematoxylin (rac-hematoxylin) comprises a mixture of (+)-hematoxylin and (-)-hematoxylin. TIFF2025114591000001.tif56170

[0048] In some embodiments, the ratio of (+)-hematoxylin to (-)-hematoxylin ranges from about 1:10 to about 10:1. In other embodiments, the ratio of (+)-hematoxylin to (-)-hematoxylin ranges from about 1:5 to about 5:1. In still other embodiments, the ratio of (+)-hematoxylin to (-)-hematoxylin ranges from about 1:4 to about 4:1. In further embodiments, the ratio of (+)-hematoxylin to (-)-hematoxylin ranges from about 1:3 to about 3:1. In still further embodiments, the ratio of (+)-hematoxylin to (-)-hematoxylin ranges from about 1:2.5 to about 2.5:1. In yet other embodiments, the ratio of (+)-hematoxylin to (-)-hematoxylin ranges from about 1:2 to about 2:1.

[0049] In some embodiments, the racemic hematoxylin comprises between about 40% and about 60% (+)-hematoxylin by weight. In other embodiments, the racemic hematoxylin comprises between about 45% and about 55% (+)-hematoxylin by weight. In yet other embodiments, the racemic hematoxylin comprises between about 48% and about 52% (+)-hematoxylin by weight. In further embodiments, the racemic hematoxylin comprises about 50% (+)-hematoxylin by weight.

[0050] In some embodiments, racemic hematoxylin can be derived according to the procedure described herein in Example 2. In other embodiments, racemic hematoxylin is as described in Dann O, Hofmann H (1965) Chromane XVII. Die Synthese von (±)-Haematoxylin. Chem. Ber. 98: 1498-1504, [German], the entire disclosure of which is incorporated herein by reference.

[0051] stabilizers Suitable stabilizers include polysaccharides such as amylose, cyclodextrins, and other cyclic or helical compounds containing multiple aldose rings, e.g., compounds formed by 1,4- and 1,6-bonds between monosaccharides (such as glucose, fructose, and galactose) and disaccharides (such as sucrose, maltose, and lactose). Other stabilizers include cryptands, cryptophanes, cavitands, crown ethers, dendrimers, nanotubes, calixarenes, valinomycin, and nigericin.

[0052] In some embodiments, stabilizers may 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, stabilizers include amylose derivatives and cyclodextrin derivatives in which one or more of the hydroxyl groups or hydrogen atoms of the hydroxyl groups of the constituent aldose rings are substituted with a substituent. 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), halogen groups (-F, -Cl, -Br, and -I), nitro groups, phosphorus-containing groups (such as phosphate groups and alkylphosphate groups), sulfur-containing groups (such as sulfate and sulfate ester groups), bridging groups (e.g., groups that link two or more hydroxyl positions on a cyclodextrin ring or link two or more stabilizers), aldehyde groups, ketone groups, oxime groups, carboxylic acid groups and derivatives thereof, carbonate groups and carbamate groups, silicon-containing groups, boron-containing groups, tin-containing groups, and hydroxyalkyl groups (such as hydroxyethyl and hydroxypropyl groups).

[0053] Specific examples of cyclodextrins include α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, and δ-cyclodextrin, as well as derivatives of each of these classes of cyclodextrin. Specific 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, cyclodextrin, carboxymethylated gamma-cyclodextrin, carboxyethylated alpha-cyclodextrin, carboxyethylated beta-cyclodextrin, carboxyethylated gamma-cyclodextrin, octyl succinated alpha-cyclodextrin, octyl succinated beta-cyclodextrin, octyl succinated gamma-cyclodextrin, acetylated alpha-cyclodextrin, acetylated beta-cyclodextrin, acetylated gamma-cyclodextrin, sulfated alpha-cyclodextrin, sulfated beta-cyclodextrin, and sulfated gamma-cyclodextrin.Other specific examples of cyclodextrin derivatives include the following β-cyclodextrin derivatives: 2,3-dimethyl-6-aminomethyl-α-cyclodextrin, 6-azido-α-cyclodextrin, 6-bromo-β-cyclodextrin, 6A,6B-dibromo-β-cyclodextrin, 6A,6B-diiodo-β-cyclodextrin, 6-O-maltosyl-β-cyclodextrin, 6-iodo-α-cyclodextrin, 6-tosyl-β-cyclodextrin, Cyclodextrin, peracetyl-maltosyl-β-cyclodextrin, 6-t-butyldimethylsilyl-β-cyclodextrin, 2,3-diacetyl-6-butyldimethylsilyl-β-cyclodextrin, 2,6-dibutyl-3-acetyl-β-cyclodextrin, 2,6-dibutyl-β-cyclodextrin, 2,6-t-butyl-dimethylsilyl-β-cyclodextrin, and 2,6-di-O-methyl-3-allyl-β-cyclodextrin. Various cyclodextrins and cyclodextrin derivatives can be obtained commercially, for example, from CTD, Inc. (High Springs, Florida, USA), or can be synthesized according to procedures outlined in the scientific literature, for example, "Synthesis of Chemically Modified Cyclodextrins," Croft and Bartsch, Tetrahedron, 39: 1417-1474, 1983.

[0054] In some embodiments, the stabilizer is one or more of a cyclodextrin or a cyclodextrin derivative. In other embodiments, the stabilizer is one or more of a β-cyclodextrin and a β-cyclodextrin derivative. In still other embodiments, the stabilizer can be α-amylose, β-amylose, or γ-amylose.

[0055] In some embodiments, stabilizers can be included in any racemic hematoxylin formulation at a concentration ranging from about 1 mM to about 1 M, from about 5 mM to about 500 mM, or from about 5 mM to about 25 mM.

[0056] In yet other embodiments, the stabilizer can be a cyclodextrin or cyclodextrin derivative, and more particularly, the stabilizer can be a cyclodextrin or cyclodextrin derivative that exhibits an aqueous solubility at about 25° C. of greater than about 5 mg / mL, e.g., greater than about 20 mg / mL, greater than about 100 mg / mL, or greater than about 500 mg / mL.

[0057] solvent A variety of solvents can be utilized in the racemic hematoxylin formulations of the present disclosure. In some embodiments, the solvent comprises one or more of water, a lower alkanol such as ethanol, and a polyol. In other embodiments, the solvent comprises an aqueous solvent comprising water and a polyol.

[0058] Suitable examples of polyols include glycerol, ethylene glycol, propylene glycol, poly(ethylene glycol), and poly(propylene glycol). In some embodiments, the aqueous solvent formulation comprises between about 5% and about 45% by volume of one or more of ethylene glycol and propylene glycol. In other embodiments, the aqueous solvent formulation comprises between about 10% and about 40% by volume of one or more of ethylene glycol and propylene glycol. In still other embodiments, the aqueous solvent formulation comprises between about 15% and about 30% by volume of one or more of ethylene glycol and propylene glycol.

[0059] antioxidants Examples of antioxidants suitable for use in any racemic hematoxylin formulation include hydroquinone, gallic acid, reducing 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 these acids (e.g., Mg 2+ , NH4 + , Na + , K. + and Ca 2+In another embodiment, the water-soluble antioxidant includes one or more of hydroquinone and n-propyl gallate.

[0060] oxidizing agent Suitable chemical oxidizing agents include molecular oxygen, naturally occurring in the atmosphere, which diffuses into and oxidizes racemic hematoxylin, and "chemical oxidizing agents" that actively combine with racemic hematoxylin (usually in solution) to convert at least a portion of the racemic hematoxylin to hematein. A semi-oxidizing hematoxylin solution is a solution containing an oxidizing agent in an amount that oxidizes approximately half of the available racemic hematoxylin, as described in Gill, Acta Cytologica, 18(4):300-11 (1974), the entire disclosure of which is incorporated herein by reference. Examples of useful chemical oxidizing agents include one or more of iodates (such as sodium iodate and potassium iodate), mercuric oxide, permanganates (such as potassium permanganate), periodates (such as sodium periodate and potassium periodate), and peroxides (such as hydrogen peroxide). In certain embodiments, the chemical oxidizing agent comprises sodium iodate.

[0061] The oxidizing agent can be present in an amount sufficient to completely (e.g., substantially quantitatively) oxidize the racemic hematoxylin to hematein, or in an amount sufficient to only partially oxidize the racemic hematoxylin to hematein. In certain embodiments, more than half of the racemic hematoxylin is oxidized to hematein by the chemical oxidizing agent, while in others, less than half of the racemic hematoxylin is oxidized to hematein by the chemical oxidizing agent. For example, between 1% and 50% of the racemic hematoxylin can be oxidized to hematein by the chemical oxidizing agent, but more typically, between about 10% and about 30% of the racemic hematoxylin is oxidized to hematein by the chemical oxidizing agent. In certain examples, the molar ratio of racemic hematoxylin to oxidizing agent used in the formulation is between 6:1 and 1:1. While the chemical oxidizing agent is considered part of the formulation, it is understood that it is converted to its reduction products upon reaction with the racemic hematoxylin. These reduction products remain in the formulation.

[0062] mordant Mordants suitable for use in any racemic hematoxylin formulation include aluminum mordants, iron mordants, bismuth mordants, copper mordants, molybdenum mordants, vanadium mordants, and zirconium mordants. In some embodiments, the mordant comprises alum. In other embodiments, the mordant comprises aluminum sulfate. In some embodiments, the mordant may be present in the formulation at a concentration higher than the concentration of hematein in the formulation (as can be determined by refractometry, thin-layer chromatography, or spectroscopy) or at a concentration lower than the concentration of hematein in the formulation. Alternatively, in some embodiments, the molar ratio of racemic hematoxylin to mordant in the formulation ranges from about 2:1 to about 1:100. In other embodiments, the molar ratio of racemic hematoxylin to mordant in the formulation ranges from about 2:1 to about 1:50. In yet other embodiments, the molar ratio of racemic hematoxylin to mordant in the formulation ranges from between about 1:5 to about 1:20.

[0063] acid In some embodiments, the racemic hematoxylin formulation may further comprise an acid and / or a buffer. Any acid can be utilized in the racemic hematoxylin formulation. Non-limiting examples of suitable acids include acetic acid, hydroiodic acid, hydrobromic acid, hydrochloric acid, nitric acid, hydrofluoric acid, nitrous acid, and formic acid.

[0064] Examples of racemic hematoxylin preparations In some embodiments, the racemic hematoxylin in the racemic hematoxylin formulation has a molar concentration between about 0.01 M and about 0.05 M. In other embodiments, the racemic hematoxylin in the racemic hematoxylin formulation has a molar concentration between about 0.02 M and about 0.04 M. In yet other embodiments, the racemic hematoxylin in the racemic hematoxylin formulation has a molar concentration of about 0.03 M.

[0065] In some embodiments, the racemic hematoxylin formulation comprises a mixture of water and ethylene glycol as a solvent, sodium iodate as an oxidizing agent, aluminum sulfate as a mordant, and β-cyclodextrin or a derivative thereof as a stabilizer. One or more water-soluble antioxidants, such as hydroquinone and n-propyl gallate, may also be included in certain such embodiments. In other embodiments, the mixture of water and ethylene glycol comprises about 10% to about 40% ethylene glycol by volume and about 60% to about 90% water by volume. In some embodiments, the racemic hematoxylin formulation comprises a mixture of water, a glycol ether, and racemic hematoxylin.

[0066] Method for preparing racemic hematoxylin preparations The present disclosure also relates to methods for preparing racemic hematoxylin formulations, such as for histochemical staining of biological samples. In some embodiments, the methods include forming a hematein solution, adding a mordant to the hematein solution to form a staining solution, and adding a stabilizer and / or an antioxidant to the staining solution to form the racemic hematoxylin formulation. In some embodiments, forming the hematein solution includes dissolving racemic hematoxylin in a solvent and adding a chemical oxidizing agent in an amount sufficient to convert at least a portion of the racemic hematoxylin to hematein. In some embodiments, the solvent used to dissolve the racemic hematoxylin comprises an aqueous composition, such as a composition comprising water and a polyol. Non-limiting examples of suitable polyols described herein include glycerol, ethylene glycol, and propylene glycol. Additional methods for forming hematoxylin compositions are described in U.S. Patent Application Publication No. 2017 / 0284908, the entire disclosure of which is incorporated herein by reference.

[0067] pH-reduced racemic hematoxylin preparation The present disclosure provides a pH-reduced hematoxylin formulation. Generally, the pH-reduced hematoxylin formulation comprises racemic hematoxylin, an acid, and at least one additive, such as any additive described herein. In some embodiments, at least two additives are included. In other embodiments, at least three additives are included. In some embodiments, the pH-reduced hematoxylin formulation comprises a solvent, racemic hematoxylin, and an acid. In some embodiments, the acid is a strong acid. In some embodiments, the strong acid is selected from hydroiodic acid, hydrobromic acid, hydrochloric acid, and nitric acid. In other embodiments, the acid is a weak acid. In some embodiments, the weak acid is selected from hydrofluoric acid and formic acid. In some embodiments, the pH-reduced hematoxylin formulation comprises racemic hematoxylin, an acid, and at least one of a mordant, an oxidizing agent, a shelf-life extender, an antioxidant, and a stabilizer.

[0068] In some embodiments, the pH-reduced hematoxylin formulation has a pH value of less than about 2.4. In some embodiments, the pH-reduced hematoxylin formulation has a pH value of less than about 2.375. In other embodiments, the pH-reduced hematoxylin formulation has a pH value of less than about 2.35. In other embodiments, the pH-reduced hematoxylin formulation has a pH value of less than about 2.325. In other embodiments, the pH-reduced hematoxylin formulation has a pH value of less than about 2.3. In other embodiments, the pH-reduced hematoxylin formulation has a pH value of less than about 2.275. In other embodiments, the pH-reduced hematoxylin formulation has a pH value of less than about 2.25. In other embodiments, the pH-reduced hematoxylin formulation has a pH value of less than about 2.225. In other embodiments, the pH-reduced hematoxylin formulation has a pH value of less than about 2.2. In other embodiments, the pH-reduced hematoxylin formulation has a pH value of less than about 2.175. In other embodiments, the reduced pH hematoxylin formulation has a pH value of less than about 2.15. In other embodiments, the reduced pH hematoxylin formulation has a pH value of less than about 2.125. In other embodiments, the reduced pH hematoxylin formulation has a pH value of less than about 2.1.

[0069] In some embodiments, the pH-reduced hematoxylin formulation has a pH value between about 1.5 and about 2.4. In other embodiments, the pH-reduced hematoxylin formulation has a pH value between about 1.5 and about 2.3. In other embodiments, the pH-reduced hematoxylin formulation has a pH value between about 1.6 and about 2.3. In other embodiments, the pH-reduced hematoxylin formulation has a pH value between about 1.8 and about 2.3. In other embodiments, the pH-reduced hematoxylin formulation has a pH value between about 2 and about 2.3. In other embodiments, the pH-reduced hematoxylin formulation has a pH value between about 2 and about 2.2. In other embodiments, the pH-reduced hematoxylin formulation has a pH value between about 1.5 and about 2.2. In other embodiments, the pH-reduced hematoxylin formulation has a pH value between about 1.6 and about 2.2.

[0070] Those skilled in the art will recognize that the amount of acid in a formulation may, of course, vary depending on the particular acid selected, the molarity of the acid, the normality of the acid, and / or the presence of other components in the racemic hematoxylin formulation. In some embodiments, the amount of acid in any pH-reduced racemic hematoxylin formulation ranges from about 0.05% to about 15% of the total volume of the formulation. In other embodiments, the amount of acid in any pH-reduced racemic hematoxylin formulation ranges from about 0.05% to about 12% of the total volume of the formulation. In still other embodiments, the amount of acid in any pH-reduced racemic hematoxylin formulation ranges from about 0.1% to about 10% of the total volume of the formulation. In still other embodiments, the amount of acid in any pH-reduced racemic hematoxylin formulation ranges from about 0.1% to about 7.5% of the total volume of the formulation. In still other embodiments, the amount of acid in any pH-reduced racemic hematoxylin formulation ranges from about 0.1% to about 6% of the total volume of the formulation. In yet other embodiments, the amount of acid in any pH-reduced racemic hematoxylin formulation ranges from about 0.1% to about 5% of the total volume of the formulation. In yet other embodiments, the amount of acid in any pH-reduced racemic hematoxylin formulation ranges from about 0.1% to about 4% of the total volume of the formulation. In yet other embodiments, the amount of acid in any pH-reduced racemic hematoxylin formulation ranges from about 0.2% to about 4% of the total volume of the formulation.

[0071] In other embodiments, the amount of acid in any pH-reduced racemic hematoxylin formulation ranges from about 0.5% to about 12% of the total volume of the formulation. In yet other embodiments, the amount of acid in any pH-reduced racemic hematoxylin formulation ranges from about 0.5% to about 10% of the total volume of the formulation. In yet other embodiments, the amount of acid in any pH-reduced racemic hematoxylin formulation ranges from about 0.5% to about 7.5% of the total volume of the formulation. In yet other embodiments, the amount of acid in any pH-reduced racemic hematoxylin formulation ranges from about 0.5% to about 6% of the total volume of the formulation. In yet other embodiments, the amount of acid in any pH-reduced racemic hematoxylin formulation ranges from about 0.5% to about 5% of the total volume of the formulation. In yet other embodiments, the amount of acid in any pH-reduced racemic hematoxylin formulation ranges from about 0.5% to about 4% of the total volume of the formulation. In yet other embodiments, the amount of acid in any pH-reduced racemic hematoxylin formulation ranges from about 1% to about 4% of the total volume of the formulation.

[0072] Method for preparing pH-reduced racemic hematoxylin preparations Some aspects of the present disclosure are directed to methods of making pH-reduced racemic hematoxylin formulations. In some embodiments, the pH-reduced racemic hematoxylin formulations can be formulated by lowering the pH of a racemic hematoxylin formulation (e.g., any of the racemic hematoxylin formulations disclosed herein; or any hematoxylin formulation comprising racemic hematoxylin and at least one other additive, including any of the above).

[0073] Generally, the pH value of a racemic hematoxylin solution can be lowered by adding an acid (e.g., a strong acid) to the racemic hematoxylin formulation. In some embodiments, the acid is a strong acid. In some embodiments, the strong acid is selected from hydrochloric acid, sulfuric acid, perchloric acid, nitric acid, or a mixture thereof. In other embodiments, the strong acid is hydrochloric acid. In some embodiments, the strong acid is 1 M hydrochloric acid. In other embodiments, the strong acid is 0.5 M hydrochloric acid. In other embodiments, the strong acid is 0.5 M sulfuric acid. In yet other embodiments, the strong acid is 0.25 M sulfuric acid. Of course, one of skill in the art could select any acid that allows the racemic hematoxylin formulation to achieve the desired pH value.

[0074] In some embodiments, an acidifying solution is added to racemic hematoxylin or a racemic hematoxylin formulation to provide a racemic hematoxylin formulation with a reduced pH. In some embodiments, the acidifying solution comprises an acid-base and one or more additives. In some embodiments, the one or more additives of the acidifying solution are selected from one or more of a stabilizer, a mordant, an oxidizing agent, a shelf-life extender, and an antioxidant. The additives of the acidifying solution can be present in the same amounts or ratios as those described herein for the racemic hematoxylin formulations described herein.

[0075] Those skilled in the art will recognize that the amount of acid and / or buffer added to the racemic hematoxylin or racemic hematoxylin formulation may, of course, vary depending on the particular acid selected, the molarity of the acid, the normality of the acid, and / or the presence of other components in the starting racemic hematoxylin formulation.

[0076] In some embodiments, the amount of acid (acidifying solution) added is such that the pH value of the racemic hematoxylin solution or formulation is reduced by about 2% to about 20%, i.e., the pH value of the initial racemic hematoxylin solution or formulation is reduced by about 2% to about 20%, providing a pH-reduced racemic hematoxylin solution or formulation having a relatively low pH. In other embodiments, the amount of acid added is such that the pH value of the racemic hematoxylin solution or formulation is reduced by about 2.5% to about 15%. In yet other embodiments, the amount of acid added is such that the pH value of the racemic hematoxylin solution or formulation is reduced by about 3% to about 12.5%. In further embodiments, the amount of acid added is such that the pH value of the racemic hematoxylin solution or formulation is reduced by about 4% to about 12%. In yet another embodiment, the acid is added in an amount such that the pH value of the racemic hematoxylin solution or racemic hematoxylin formulation is reduced by about 5% to about 10%.

[0077] In some embodiments, the change in pH value (i.e., decrease in pH value) of the racemic hematoxylin solution or racemic hematoxylin formulation upon addition of acid (or acidifying solution) is about 0.25 (i.e., a decrease of about 0.25 pH units). In other embodiments, the change in pH value of the racemic hematoxylin solution or racemic hematoxylin formulation upon addition of acid is about 0.225. In other embodiments, the change in pH value of the racemic hematoxylin solution or racemic hematoxylin formulation upon addition of acid is about 0.2. In some embodiments, the change in pH value of the racemic hematoxylin solution or racemic hematoxylin formulation upon addition of acid is about 0.175. In other embodiments, the change in pH value of the racemic hematoxylin solution or racemic hematoxylin formulation upon addition of acid is about 0.15. In another embodiment, the change in pH value of the racemic hematoxylin solution or formulation upon addition of acid is about 0.125. In another embodiment, the change in pH value of the racemic hematoxylin solution or formulation upon addition of acid is about 0.1. In another embodiment, the change in pH value of the racemic hematoxylin solution or formulation upon addition of acid is about 0.075.

[0078] In some embodiments, an amount of acid (or acidifying solution) is added to the racemic hematoxylin solution or racemic hematoxylin formulation to lower the pH value of the formulation to less than about 2.4. In other embodiments, an amount of acid is added to the racemic hematoxylin solution or racemic hematoxylin formulation to lower the pH value of the formulation to less than about 2.35. In other embodiments, an amount of acid is added to the racemic hematoxylin solution or racemic hematoxylin formulation to lower the pH value of the formulation to less than about 2.3. In other embodiments, an amount of acid is added to the racemic hematoxylin solution or racemic hematoxylin formulation to lower the pH value of the formulation to less than about 2.25. In other embodiments, an amount of acid is added to the racemic hematoxylin solution or racemic hematoxylin formulation to lower the pH value of the formulation to less than about 2.2. In other embodiments, an amount of acid is added to the racemic hematoxylin solution or racemic hematoxylin formulation such that the pH value of the formulation is lowered to less than about 2.15, hi other embodiments, an amount of acid is added to the racemic hematoxylin solution or racemic hematoxylin formulation such that the pH value of the formulation is lowered to less than about 2.1.

[0079] Regardless of the nature of the starting hematoxylin solution selected, one skilled in the art can add an appropriate amount of acid (or acidifying solution) to lower the pH value of the racemic hematoxylin solution or racemic hematoxylin preparation.

[0080] Method for readjusting the pH value of pH-decreased racemic hematoxylin preparations

[0081] Some aspects of the present disclosure are directed to readjusting the pH value of a pH-reduced racemic hematoxylin formulation to provide a pH-adjusted racemic hematoxylin formulation suitable for staining. In some embodiments, the method includes adding a readjustment solution to the pH-reduced racemic hematoxylin formulation. In some embodiments, the readjustment solution includes a solvent and one of a strong base and / or a buffer. In some embodiments, the solvent is selected from those enumerated herein. In some embodiments, the same solvent is used in the pH-reduced racemic hematoxylin formulation and the readjustment solution.

[0082] In some embodiments, the strong base is selected from the group consisting of sodium hydroxide, potassium hydroxide, and lithium hydroxide. In other embodiments, the base is ammonia. In some embodiments, the strong base is 1 M sodium hydroxide. In other embodiments, the strong base is 0.5 M sodium hydroxide. In yet other embodiments, the strong base is 0.1 M sodium hydroxide.

[0083] In some embodiments, the buffer is selected from maleate, phosphate, glycine, citrate, glycylglycine, malate, formate, cyanoacetate, succinate, acetate, propionate, fumarate, sulfate, alanine, arginine, isoleucine, leucine, norleucine, proline, serine, threonine, or any combination thereof. In some embodiments, the weak acid of the buffer has a pK ranging from about 1.5 to about 3.5. a In other embodiments, the weak acid of the buffer has a pK in the range of about 1.8 to about 3.2. a In yet other embodiments, the weak acid of the buffer has a pK in the range of about 2 to about 3. a It has.

[0084] In some embodiments, the reconditioning solution further comprises an additive selected from the group consisting of a mordant, an oxidizing agent, a shelf life extender, and an antioxidant.

[0085] In some embodiments, a sufficient amount of strong base, buffer solution, or readjustment solution is added to the pH-reduced racemic hematoxylin formulation to raise the pH value of the pH-reduced racemic hematoxylin formulation to at least about 2.4. In other embodiments, the amount of strong base, buffer solution, or readjustment solution is added to raise the pH of the pH-reduced racemic hematoxylin formulation to at least about 2.45. In yet other embodiments, the amount of strong base, buffer solution, or readjustment solution is added to raise the pH of the pH-reduced racemic hematoxylin formulation to at least about 2.5. In further embodiments, the amount of strong base, buffer solution, or readjustment solution is added to raise the pH of the pH-reduced racemic hematoxylin formulation to at least about 2.55.

[0086] In some embodiments, the method comprises measuring the initial pH value of an aliquot of the pH-reduced racemic hematoxylin formulation and adding an amount of strong base, buffer solution, or readjustment solution until the pH value of the aliquot is raised to at least about 2.4. In some embodiments, the method comprises measuring the initial pH value of an aliquot of the pH-reduced racemic hematoxylin formulation and adding an amount of strong base, buffer solution, or readjustment solution until the pH value of the aliquot is raised to at least about 2.45. In some embodiments, the method comprises measuring the initial pH value of an aliquot of the pH-reduced racemic hematoxylin formulation and adding an amount of strong base, buffer solution, or readjustment solution until the pH value of the aliquot is raised to at least about 2.5. In some embodiments, the method comprises measuring the initial pH value of an aliquot of the pH-reduced racemic hematoxylin formulation and adding an amount of strong base, buffer solution, or readjustment solution until the pH value of the aliquot is raised to at least about 2.55.

[0087] System for hematoxylin staining In some embodiments, the racemic hematoxylin formulations described herein are applied or introduced to the biological sample manually or using a dip-and-dunk technique. In other embodiments, the racemic hematoxylin formulation is applied or dispensed to the biological sample, such as by an automated staining device. Those skilled in the art will recognize that dispensing any racemic hematoxylin formulation refers to applying the racemic hematoxylin formulation to a biological sample or substrate (e.g., a slide).

[0088] The methods and formulations disclosed herein can be adapted for use with existing automated processing systems. For example, Ventana Medical Systems, Inc. is the assignee of numerous U.S. patents disclosing systems and methods for performing automated analyses, including U.S. Patent Nos. 5,650,327, 5,654,200, 6,296,809, 6,352,861, 6,827,901, and 6,943,029, and U.S. Patent Application Publication Nos. 20030211630 and 20040052685, each of which is incorporated herein by reference. These systems can be adapted to be compatible with the present invention. Briefly, the automated slide processing system described in the aforementioned applications is a high-volume slide processing system that shuttles trays holding multiple slides in a substantially horizontal position (to minimize cross-contamination) between workstations where various slide processing operations are performed on the slides. Fresh reagents can be applied to each slide during processing, and slides are spaced apart and processed separately in the tray, substantially eliminating cross-contamination of slides with reagents. In one configuration, the system includes a radiant heater, a combination deparaffinizer / stainer / solvent exchange workstation, a convection oven, and a coverslipper. Trays of slides carrying paraffin-embedded tissue samples can be heated under the system's radiant heater to spread the paraffin within the samples and facilitate removal, as well as to adhere the samples to the slides. The trays can then be transported to the multifunctional deparaffinizer / stainer / solvent exchange workstation, where the slides can be deparaffinized, stained, and solvent exchanged. Trays of stained slides ready for coverslipping can then be shuttled to and from the system's coverslipper, which adds coverslips to the slides. Once the slides are coverslipped, the trays can be transported to a convection oven to harden the coverslips on the stained slides. The high-volume stainer just described is commercially available from Ventana Medical Systems, Inc., Tucson, Arizona, USA.

[0089] Examples of other commercially available specimen processing systems to which the solutions and formulations described herein can be applied include the VENTANA SYMPHONY (individual slide stain) and VENTANA HE600 (individual slide stain) systems, as well as the Dako CoverStainer (batch stain), Leica ST4020 Small Linear Stainer (batch stain), Leica ST5020 Multistainer (batch stain), and Leica ST5010 Autostainer XL systems (batch stain) from Agilent Technologies, and H&E stainers from Leica Biosystems Nussloch GmbH.

[0090] In some embodiments, the staining system described above can be configured to perform any histological staining process, but an exemplary hematoxylin-eosin staining protocol includes a baking step to adhere the sample to the slide, a deparaffinization step to remove paraffin from paraffin-embedded samples, a hematoxylin staining step (which can utilize the disclosed hematoxylin formulations), a bluing step in which the pH is increased to turn the hematoxylin blue and provide improved contrast with the eosin added downstream, an eosin staining step, a differentiation step used to remove excess eosin and change the eosin to various shades of red to pink, a dehydration step using absolute ethanol to remove water from the sample, a step in which the slide is exposed to elevated temperature and airflow to remove hydrocarbons, a coverslip step to distribute D-limonene onto the sample, and a curing step.

[0091] While the staining system described above can be configured to perform any histological staining process, an exemplary hematoxylin-eosin staining protocol includes a baking step to adhere the sample to the slide, a deparaffinization step to remove paraffin from paraffin-embedded samples, a hematoxylin staining step (which can utilize the disclosed hematoxylin formulations), a bluing step in which the pH is increased to turn the hematoxylin blue and enhance contrast with the eosin added downstream, an eosin staining step, a differentiation step used to remove excess eosin and change the eosin to various shades of red to pink, a dehydration step in which water is removed from the sample using a glycol ether (e.g., dipropylene glycol propyl ether), a step in which the slide is exposed to elevated temperature and airflow to remove hydrocarbons, a coverslip step in which D-limonene is distributed onto the sample, and a curing step.

[0092] In some embodiments, the automated staining system includes one or more reservoirs, containers, and / or dispensers containing the racemic hematoxylin formulation. In other embodiments, the automated staining system includes separate reservoirs or containers containing the pH-reduced hematoxylin formulation and the reconditioning solution. In some embodiments, the system further includes a dispensing system that delivers the racemic hematoxylin formulation (after reconditioning of the pH-reduced racemic hematoxylin formulation) and any other solutions to a biological sample, e.g., a biological sample mounted on a slide. In some embodiments, the pH-reduced racemic hematoxylin formulation and / or other solutions (e.g., reconditioning solution) are combined prior to application to the biological sample. In some embodiments, the container of the pH-reduced racemic hematoxylin formulation and the container of another solution (e.g., reconditioning solution) are pressurized and fluidly connected to a mixing container. The mixing container can be any container capable of holding or transporting the mixed solution, such as rigid or flexible tubing. In some embodiments, the mixing container is tubing fluidly connected to a dispenser. In some embodiments, the pH-reduced racemic hematoxylin formulation and the readjustment solution are fluidly connected to a T-fitting via tubing. The output from the T-fitting is then fluidly connected to a dispenser. In these embodiments, the pH-reduced racemic hematoxylin formulation and the readjustment solution are supplied to the T-fitting, and mixing of the solutions occurs in the tubing extending from the T-fitting. In some embodiments, the pH-reduced racemic hematoxylin formulation and the readjustment solution are separately dispensed onto a biological sample. In these embodiments, the solutions can be mixed by diffusion on the sample or mechanically mixed, such as by stirring with a pipette.

[0093] In some embodiments, an automated specimen processing device can include a carousel that holds multiple substrates, such as microscope slides, each containing a biological sample to be stained. In some embodiments, the automated staining device can also include a device for rotating the carousel at a predetermined speed and a mechanism for directing and controlling the application of reagents, including the solutions and formulations described herein, to the substrates and samples during rotation of the carousel. In some embodiments, once slides are loaded into the device, a test protocol dictates which fluids are to be dispensed onto the substrates at specific times. In some embodiments, at the appropriate time, a dispenser rack rotates to align the correct fluids onto the substrates, and the device dispenses a predetermined amount of fluid onto the substrate. In some embodiments, the device allows the fluids to remain in contact with the biological samples for a predetermined period of time.

[0094] In some embodiments, the system is an automated slide processing system that includes a slide tray that holds a plurality of slides in a substantially horizontal position (e.g., in two rows, with the slides held at an angle between about 0.2 degrees and about 1.2 degrees from horizontal) and one or more workstations that receive the slide tray and perform one or more slide processing operations on the slides (e.g., arranged in a vertical stack) in the slide tray. In some embodiments, the workstations can perform slide processing operations on one or more individual slides in the slide tray, e.g., at least two or four slides in the slide tray, or can perform slide processing operations on all slides in the slide tray simultaneously. In some embodiments, the one or more workstations dispense reagents onto the slides in the slide tray without a substantial amount of reagent contacting a first slide that contacts a second slide, thereby minimizing cross-contamination between slides. Such a workstation can include one or more directional nozzles that dispense reagents onto the slides; for example, the one or more directional nozzles can include a pair of directional nozzles that dispense reagents in opposite directions across the surface of the slide. In more specific embodiments, the one or more directional nozzles can further include a directional nozzle that dispenses reagents toward the bottom surface of the slide. In certain other embodiments, one or more workstations can simultaneously dispense a reagent (e.g., the same reagent) to at least two slides held in a slide tray within a given workstation, or one or more workstations can simultaneously dispense a reagent (such as the same reagent) to all slides held in a slide tray within a given workstation. Additional system components and tray configurations (as well as control systems) are described in U.S. Patent Nos. 8,663,991, 7,468,161, and 9,528,918, the disclosures of which are incorporated herein by reference in their entireties.

[0095] In some embodiments, the present disclosure provides an apparatus for automatically processing biological specimens, the apparatus including at least one slide tray for holding a plurality of slides in a substantially horizontal position, with the biological specimen positioned on the slide; one or more workstations for receiving the slide tray and performing one or more slide processing operations on the plurality of slides held in the slide tray; a transporter for transporting the slide tray into and out of the one or more workstations; a fluidics module in fluid communication with the one or more workstations and supplying reagents to the one or more workstations; a pneumatic module in fluid communication with the one or more workstations and the fluidics module, the pneumatic module providing vacuum and / or pressurized gas to the one or more workstations and the fluidics module; and a control module in electrical communication with the transporter, the one or more workstations, the fluidics module, and the pneumatic module, the control module coordinating the function of the apparatus components during processing of the biological specimens. The apparatus may be adapted to deliver one or more of the solutions and / or formulations described herein.

[0096] Counterstaining In some embodiments, the systems and methods further include staining the biological sample with an additional stain, such as a counterstain. In some embodiments, contacting the sample with the counterstain includes contacting the sample with one or more of Eosin Y (CAS No. 15086-94-9), Orange G (CAS No. 1936-15-8), Light Green SF Yellow (CAS No. 5141-20-8), Bismarck Brown (CAS No. 8005-77-4), Fast Green FCF (CAS No. 2353-45-9), OG-6 (including Orange G), EA25 (including Light Green SF, Bismarck Brown, and Eosin Y), EA36 (including Light Green SF, Bismarck Brown, and Eosin Y), EA50 (including Light Green SF, Bismarck Brown, and Eosin Y), and EA65 (including Light Green SF, Bismarck Brown, and Eosin Y). Formulas and methods for preparing such counterstains can be found, for example, in StainsFile (an internet resource for histotechnologists maintained by Bryan Llewellyn); Kiernan, "Histological and Histochemical Methods: Theory and Practice," 3rd Ed. Butterworth Heinemann, Oxford, UK; and Horobin and Kiernan, "Conn's Biological Stains: A Handbook of Dyes, Stains, and Fluorochromes for Us in Biology and Medicine," 10th ed., Oxford: BIOS, ISBN 1859960995, 2002. In other embodiments, contacting the sample with the hematoxylin preparation comprises a progressive hematoxylin staining protocol. In other embodiments, contacting the sample with the hematoxylin preparation comprises a regressive hematoxylin staining protocol. The method can be automated and can be performed on a biological sample supported on a substrate, such as a microscope slide.In certain embodiments, the method is used to stain tissue sections or cytological samples mounted on microscope slides. In certain embodiments further comprising a counterstaining step, the method may be a hematoxylin-eosin or PAP staining method, more particularly an automated hematoxylin-eosin or PAP staining method.

[0097] Other histological stains useful in connection with the staining procedures of the present invention include dyes such as acridine dyes, anthraquinone dyes, arylmethane dyes, azo dyes, diazonium dyes, nitro dyes, phthalocyanine dyes, quinine imine dyes, tetrazolium dyes, thiazole dyes, and xanthene dyes. Examples of dyes useful for histological staining include acetyl yellow, acid black 1, acid blue 22, acid blue 93, acid fuchsin, acid green, acid green 1, acid green 5, acid magenta, acid orange 10, acid red 4, acid red 26, acid red 29, acid red 44, acid red 51, acid red 66, acid red 73, acid red 87, acid red 91, acid red 92, acid red 93, acid red 94, acid red 95, acid red 96, acid red 97, acid red 98, acid red 99, acid red 100, acid red 101, acid red 102, acid red 103, acid red 104, acid red 105, acid red 106, acid red 107, acid red 108, acid red 109, acid red 1109, acid red 1111, acid red 112, acid red 113, acid red 114, acid red 115, acid red 116, acid red 117, acid red 118, acid red 119, acid red 119, acid red 119, acid red 119, acid red 119, acid red 120, acid red 121, acid red 122, acid red 123, acid red 124, acid red 125, acid red 126, acid red 127, acid red 128, acid red 129, acid red 129, acid Dread 94, Acid Red 101, Acid Red 103, Acid Rosein, Acid Rubin, Acid Violet 19, Acid Yellow 1, Acid Yellow 9, Acid Yellow 23, Acid Yellow 24, Acid Yellow 36, Acid Yellow 73, Acid Yellow S, Acid Yellow T, Acridine Orange, Acriflavine, Alcian Blue, Alcian Yellow, Alcohol-soluble Eosin, Alizarin, Alizarin Blue, Alizarin Blue Rue 2RC, Alizarin Carmine, Alizarin Cyanine BBS, Alizarol Cyanine R, Alizarin Red S, Alizarin Purpurin, Aluminon, Amido Black 10B, Amidonaphthol Red, Amido Schwarz, Aniline Blue WS, Aniline Purple, Anthracene Blue SWR, Anthracene Blue SWX, Auramine 0, Azoeosin, Azocarmine B, Azocarmine G, Azoeosin G, Azoic Diazo 5, Azoic Diazo 48, Azophloxine, Azoban Blue, Azure A, Azure B, Azure C, Basic Blue 8, Basic Blue 9, Basic Blue 12, Basic Blue 15, Basic Blue 17, Basic Blue 20, Basic Blue 26, Basic Brown 1, Basic Fuchsin, Basic Green 4, Basic Green 5, Basic Orange 14, Basic Red 2, Basic Red 5, Basic Red 9, Basic Violet 2, Basic Violet 4Basic Violet 10, Basic Violet 14, Basic Yellow 1, Basic Yellow 2, Biebrich Scarlet, Biebrich Scarlet R, Bismarck Brown Y, Braziline, Brazilin, Brilliant Crocein, Brilliant Crystal Scarlet 6R, Calcium Red, Carmine, Carminic Acid Carmoisine 6R, Celestine Blue B, China Blue, Chloranthine Fast Red 5B, Cochineal, Celestine Blue, Chicago Blue 4B, Chrome Violet CG, Chromotrope 2R, Chromoxantha Nin R, Congo Corinth, Congo Red, Cotton Blue Cotton Red, Black Scarlet, Black Scarlet 3B, Black Scarlet MOO, Crocin, Crystal Ponceau 6R, Crystal Scarlet, Crystal Violet, Dahlia, Diamond Green B, Direct Blue 14, Direct Blue 58, Direct Red, Direct Red 10, Direct Red 28, Direct Red 80, Direct Red 81, Direct Yellow 7, Durazol Blue 4R, Durazol Blue 8G, Eosin B, Eosin Thin Blueish, Eosin, Eosin Y, Eosin Yellowish, Eosinol, Erie Garnet B, Eriochrome Cyanine R, Erythrosin B, Ethyl Eosin, Ethyl Green, Ethyl Violet, Evans Blue, Fast Blue B, Fast Green FCF, Fast Red B, Fast Yellow, Fast Yellow Extra, Fast Yellow G, Fat Black HB, Fluorescein, Food Green 3, Galleon, Gallamine Blue, Gallocyanine, Gentian Violet, Heliofast Rubin BBL, Helvetia Blue , Hoffmann Violet, Hydrazine Yellow, Imperial Red, Ingrain Blue 1, Ingrain Yellow 1, INT, Kermes, Kermes Acid, Kern Echtrot, Lac, Laccaic Acid, Lauss Violet, Light Green, Lissamine Fast Yellow, Lissamine Green SF, Luxol Fast Blue, Magenta 0, Magenta I, Magenta II, Magenta III, Malachite Green, Manchester Brown, Martilus Yellow, Mauve, Mauveine, Merbromin, Mercurochrome, Metanil Yellow, Methylene Azure A,Methylene Azure B, Methylene Azure C, Methylene Blue, Methylene Green, Methyl Blue, Methyl Green, Methyl Violet, Methyl Violet 2B, Methyl Violet 10B, Milling Yellow 3G, Mordant Blue 3, Mordant Blue 10, Mordant Blue 14, Mordant Blue 23, Mordant Blue 32, Mordant Blue 45, Mordant Red 3, Mordant Red 11, Mordant Violet 25, Mordant Violet 39, Naphthalene Blue Black, Naphthol Blue Black, Naphthol Green B, Naphthol Yellow S, Natural Black 1, Natural Red, Natural Red 3, Natural Red 4, Natural Red 8, Natural Red 16, Natural Red 24, Natural Red 25, Natural Red 28, Natural Yellow 6, NBT, Neutral Red, New Fuchsin, Niagara Blue 3B, Night Blue, Nile Blue, Nile Blue A, Nile Blue Sulfate, Nile Red, Nitro BT, Nitro Blue Tetrazolium, Nuclear Fast Red, Oil Red 0, Orange G, Orcein, Pararosaniline, Parkinson's disease Violet, Phloxine B, Picric Acid, Ponceau 2R, Ponceau 6R, Ponceau B, Ponceau S, Pontamine Sky Blue 5B, Primula, Primulin, Purpurin, Pyronin B, Pyronin G, Pyronin Y, Rhodamine B, Rosaniline, Rose Bengal, Saffron, Safranin 0, Scarlet R, Scarlet Red, Scharlach R, Shellac, Sirius Red F3B, Sirius Red 4B, Sirius Supra Blue F3R, Solochrome Cyanine R, Soluble Blue, Solvent Black 3, Solvent Blue 38, Solvent Red 23, Solvent Red 24, Solvent Red 27, Solvent Red 45, Solvent Yellow 94, Spirit Soluble Eosin, Sudan III, Sudan IV, Sudan Black B, Sudan Red BK, Sulfur Yellow S, Swiss Blue, Tartrazine, Thioflavin S, Thioflavin T, Thionine, Toluidine Blue, Toluoyline Red, Tropaeolin G, Trypaflavin, Trypan Blue, Uranine, Vicoria Blue 4R, Victoria Blue B, Victoria Blue R, Victoria Green B,Examples include water blue I, water-soluble eosin, wood stain scarlet, and yellowish eosin, and combinations thereof. Formulas and methods for making and using the histochemical stain solutions discussed in this paragraph (such as "special staining" procedures or counterstains for specific histological situations) can be found, for example, in StainsFile (an internet resource for histotechnologists maintained by Bryan Llewellyn); Kiernan, "Histological and Histochemical Methods: Theory and Practice," 3rd Ed. Butterworth Heinemann, Oxford, UK; and Horobin and Kiernan, "Conn's Biological Stains: A Handbook of Dyes, Stains, and Fluorochromes for Us in Biology and Medicine," 10th Ed., Oxford: BIOS, ISBN 1859960995, 2002. The contents of the two combined references cited immediately above are hereby incorporated by reference. [Example]

[0098] Example 1 - Tissue staining using racemic hematoxylin preparations preface Two separate lots of VENTANA HE600 hematoxylin solution were prepared and evaluated by HPLC analysis and functional staining. One lot was prepared from commercially available hematoxylin, and the other lot was prepared from racemic hematoxylin (e.g., prepared according to the semi-synthetic method described in Example 2 herein). Both solutions were evaluated by functional testing (H&E staining) and HPLC analysis, and both solutions were found to be essentially equivalent.

[0099] reagent (1) EDTA / phenol red dilution To the bottle, add 1 L of commercially available 0.02 M EDTA solution. Add 0.01 g of phenol red sodium salt indicator dye. Mix until the dye is dissolved. (2) 25 mM ammonium formate mobile phase buffer solution (pH = 4, aqueous) Add approximately 1 L of deionized (DI) water to the bottle. Add 1.0 mL of 99% by weight formic acid. Add 1.3 mL of approximately 29% by weight aqueous ammonia solution and stir. (3) Acetonitrile (HPLC grade) (4) Diluent for MS detector To a 5 L bottle, add 500 mL of HPLC grade acetonitrile. Bring the volume to 5 L with deionized water. Add 1 mL of 99% by weight formic acid. Mix by inverting several times. (5) Hematoxylin (commercially available) (6) Hematoxylin (racemic) (7) Ethylene glycol (8) Sodium iodate (9) Aluminum sulfate hydrate (10) Hydroquinone (11) β-Cyclodextrin hydrate

[0100] Sample preparation Two samples of VENTANA HE600 Hematoxylin Solution were prepared according to the following HE600 Hematoxylin Formulation Development Method: Add 65 mL of deionized water to the container. Begin stirring at medium speed. Add 28 g of ethylene glycol. Add 0.61 g of hematoxylin. Mix moderately for 30 to 120 minutes. Add 0.065 g of sodium iodate. Within 10 minutes of adding the sodium iodate, 2.7 g of aluminum sulfate hydrate is added. Mix moderately for 30 to 120 minutes. Add 0.93g of hydroquinone. Add 1.1 g of β-cyclodextrin. Make up to 100 mL with deionized water. Mix moderately for 30 to 120 minutes. The pH value of the final solution should be between 2.45 and 2.54.

[0101] Analysis method Each HE600 hematoxylin solution was analyzed by HPLC under the following conditions: Sample preparation: Dilute 20 μL of sample to 1 mL with EDTA / phenol red diluent. Equipment: Waters Alliance e2695 Separation Module Duration: 30 minutes Injection volume: 20 μL Flow rate: 1mL / min Column ID: Waters XBridge C18 Column length: 150 mm Column diameter: 4.6 mm Particle size: 3.5μm Mobile phase A: 25 mM ammonium formate buffer, pH = 4 Mobile phase B: acetonitrile Column temperature: 40℃ UV-Vis detector: Waters 2998 photodiode array Mass detector: Waters QDa mass detector

[0102] result The chromatograms for both solutions (Figures 1A and 2A) showed similar qualitative responses (see also Figures 1B and 2B). The major peaks for hydroquinone, hydroxyhematein, hematoxylin, and hematein were all present in both chromatograms. The same unidentified minor peaks were present in both chromatograms. Each sample with quantitative results was analyzed for internal standards. All components in the two solutions showed approximately equal levels, except for the hematoxylin level. The racemic hematoxylin preparation showed approximately 1.5-fold higher hematoxylin levels compared to the sample prepared with commercial hematoxylin. The results for both chromatograms are summarized in Table 1. TIFF2025114591000002.tif42170

[0103] Functional Dyeing Eight pairs of 5-in-1 tissue slides were stained with hematoxylin and eosin (H&E) staining on a VENTANA HE600 instrument using formulations made from racemic hematoxylin and commercial hematoxylin. The only significant difference observed in the staining results was that the solution made from commercial hematoxylin (e.g., see Figures 3A and 3B) showed slightly more mucin staining than the solution made from semi-hematoxylin (e.g., see Figures 4A and 4B).

[0104] Consideration The racemic and commercial hematoxylin solutions produced HPLC chromatograms with similar qualitative results, suggesting that both hematoxylin sources were oxidized by sodium iodate to form hematein and hydroxyhemate during the formulation process. Racemic hematoxylin appeared to be chemically equivalent to commercial hematoxylin.

[0105] The higher concentration of hematoxylin in the chromatogram of the racemic hematoxylin suggested that this hematoxylin source may be purer than the commercially available source. The oxidation products of hematoxylin (hydroxyhemate and hematein) were equal in the two different solutions. If sodium iodate (the oxidizing agent) was the limiting reagent in the formulation and the starting hematoxylin concentration was higher (higher purity) in the racemic hematoxylin, the levels of these oxidation products would be equal after oxidation was complete in the two solutions, but the racemic hematoxylin solution would still have a high level of unoxidized hematoxylin.

[0106] For H&E staining on the VENTANA HE600 instrument, comparable hematoxylin solutions often show slight but significant staining differences. These differences are due to variability associated with the staining process on the VENTANA HE600 instrument. Racemic hematoxylin and commercial hematoxylin showed only slight differences in staining (mucin staining), so it cannot be said that the solutions stained differently.

[0107] The studies described in this example suggested that racemic hematoxylin and commercial hematoxylin were analytically and functionally equivalent.

[0108] Example 2 – Semisynthesis of racemic hematoxylin 1.1 General method All reactions were magnetically stirred and carried out under a positive pressure of inert gas (N or argon) using standard Schlenk techniques. Glassware was repeatedly dried in vacuo at 620 °C before use. Liquid reagents and solvents were added via syringe or oven-dried stainless steel cannulae through rubber septa. Solids were added under countercurrent inert gas or dissolved in an appropriate solvent. Low-temperature reactions were carried out in a Dewar vessel filled with coolant (acetone / dry ice (-78 °C) or H2O / ice (0 °C)). Reaction temperatures above room temperature were carried out in a heated oil bath. When literature procedures were followed, respective references are added in the experimental details. Yields refer to isolated, homogeneous, and spectroscopically pure material unless otherwise specified.

[0109] 1.1.1 Solvents and Reagents: Dry solvents such as acetone, tetrahydrofuran (THF), ethyl acetate, and toluene were purchased from commercial suppliers and used as received. Solvents for extraction and flash column chromatography were purchased in HPLC grade. 6S,11R-Hemtoxylin (1) was purchased from Sigma-Aldrich. All other reagents and solvents were purchased from chemical suppliers (Sigma-Aldrich, Acros Organics, Alfa Aesar, TCI Europe, abcr) and used as received.

[0110] 1.1.2 Chromatography: Reactions and chromatographic fractions were monitored by qualitative thin layer chromatography (TLC) on Merck KGaA silica gel F254 TLC plates. Analytes on glass plates were visualized by irradiating with ultraviolet light and / or by immersing the TLC plates in the appropriate staining solution followed by heating with a hot air gun (350°C). The following staining solutions were applied:

[0111] KMnO4 staining solution (3.0g KMnO4, 20g K2CO3, 5.0mL 5.0 wt% NaOH aqueous solution, 0.3L H2O).

[0112] Flash column chromatography was performed on Geduran® Si60 (40-63 μm) silica gel from Merck KGaA. All fractions containing the desired substrate were combined and the solvent removed under reduced pressure, followed by drying in vacuo (10 -2 mbar).

[0113] 1.1.3 NMR Spectroscopy: NMR spectra were measured on an Agilent 400-MR DD2 400 MHz spectrometer equipped with a OneNMR Probe operating at 400 MHz for proton nuclei (100 MHz for carbon nuclei). DMSO-d6 and CDCl3 were purchased from Sigma-Aldrich. 1 H NMR shifts are reported in ppm relative to the residual shift of TMS. 1 H NMR shifts were calibrated to residual solvent resonances: DMSO-d6 (2.50 ppm) and CDCl3 (7.26 ppm). 13 C NMR shifts were calibrated to the center of multiple signals of residual solvent resonances: DMSO-d6 (29.84 ppm) and CDCl3 (77.16 ppm). 1 H NMR spectroscopic data are reported as follows: chemical shift (multiplicity, coupling constant J, integrated intensity) in ppm. Multiplicities are s (singlet), br (broad signal), d (doublet), t (triplet), q (quartet), m (multiplet), and m C (centrosymmetric multiplet). In the case of complex multiplicities, the multiplicity with the smaller coupling constant is stated first. Chemical shifts of all signals except for multiplets, as well as those of centrosymmetric multiplets, are reported as the center of the resonance range. 1 H and 13 In addition to C NMR measurements, signals were assigned using 2D NMR techniques such as homonuclear correlation spectroscopy (COSY), heteronuclear single quantum coherence (HSQC), and heteronuclear multiple bond coherence (HMBC). Coupling constants, J, are reported in Hz. All NMR spectra were analyzed using the program ACD / Spectrus Processor 2015.2.7 from Advanced Chemistry Development, Inc.

[0114] 1.1.4 Mass spectrometry: Low-resolution mass spectra (LRMS) were recorded on a Waters GmbH HPLC-MS system (2695 Separation Module, 996 Photodiode Array Detector, Micromass ZQ, Grace Vydac 218TP C18 5u) computer-controlled by Waters MassLynx V4.1. For each analyte, only the characteristic molecular fragment or molecular ion peak is shown.

[0115] 1.2 Synthetic Route TIFF2025114591000003.tif75170

[0116] Scheme 1: Synthetic route to rac-hematoxylin (6) starting from commercially available natural 6S,11R-hematoxylin (1).

[0117] 1.3 Synthesis Procedure TIFF2025114591000004.tif29170

[0118] Benzyl ether 2: 6S,11R-Hemtoxylin (1, 1.00 g, 3.31 mmol, 1.00 equiv.) and K2CO3 (2.05 g, 14.9 mmol, 4.50 equiv.) were dissolved in dry acetone (33 mL) at room temperature under an inert gas atmosphere. Benzyl bromide (1.77 mL, 14.9 mmol, 4.50 equiv.) was then added in one portion, and the resulting mixture was vigorously stirred and heated to 56 °C (oil bath temperature) for 20 h. When TLC analysis indicated complete conversion of 1, the reaction was cooled to room temperature and quenched by the addition of ethyl acetate (150 mL) and brine (100 mL) to the stirred mixture. The layers were separated, and the aqueous phase was extracted with ethyl acetate (75 mL, twice). The combined organic layers were dried over Na2SO4, filtered, and concentrated in vacuo. Final purification of the crude benzyl ether 2 was achieved by flash column chromatography (silica, n-Hex: EtOAc = gradient from 80:20 to 60:40) to give 6S,11R-tetrabenzyl-hematoxylin (2, 2.01 g, 3.03 mmol, 92%) as a yellowish solid.

[0119] R f (n-Hex: EtOAc = 2:3) = 0.60.

[0120] 1 H NMR (CDCl, 400 MHz): δ = 7.46-7.27 (m, 20H), 6.92 (dd, J = 1.00, 8.66 Hz, 1H), 6.87 (d, J = 1.00, 1H), 6.80 (s, 1H), 6.69 (d, J = 8.78 Hz, 1H), 5.15 (s, 2H), 5.11 (s, 2H), 5.08 (s, 2H), 5.08 (s, 2H), 4.04 (s, 1H), 4.01 (dd, J = 1.38, 11.29 Hz, 1H), 3.72 (d, J = 11.04 Hz, 1H), 3.17 (d, J = 16.19 Hz, 1H), 2.84 (d, J = 15.43 Hz, 1H), 2.31 (s, 1H) ppm.

[0121] 13 C NMR (CDCl3, 100 MHz): δ = 151.6, 149.1, 148.5, 148.0, 137.8, 137.5, 137.4, 137.4, 137.2, 137.0, 132.0, 128.9, 128.7, 128.6, 128.6, 128.2, 128.0, 127.9, 127.7, 127.5, 127.4, 125.0, 116.9, 112.5, 112.4, 108.4, 77.52, 75.3, 72.2, 71.8, 71.5, 70.4, 50.7, 41.2 ppm. TIFF2025114591000005.tif13170TIFF2025114591000006.tif45170

[0122] Monothiocarbonate 3: 6S,11R-tetrabenzyl-hematoxylin (2, 0.50 g, 0.75 mmol, 1.00 equiv.) was dissolved in dry THF (10.0 mL) under an inert gas atmosphere at room temperature and then cooled to −78 °C. Methyllithium (0.59 mL, 0.94 mmol, 1.25 equiv.) was then added dropwise to the stirred solution. After stirring at room temperature for 30 min, the reaction was again cooled to −78 °C, and O-phenylchlorothionoformate (0.13 mL, 0.94 mmol, 1.25 equiv.) was added dropwise. The resulting mixture was stirred at room temperature for 2 h, and TLC analysis indicated complete conversion of ether 2. The reaction was quenched by adding dichloromethane (100 mL) and saturated aqueous NaHCO3 (75 mL) to the stirred mixture. The layers were separated, and the aqueous phase was extracted with ethyl acetate (75 mL, twice). The combined organic layers were dried over Na2SO4, filtered, and concentrated in vacuo (up to 30 °C). Final purification of the crude monothiocarbonate 3 was achieved by flash column chromatography (silica, n-Hex: EtOAc = 95:5 to 85:15 gradient) to afford 6S,11R-tetrabenzyl-hematoxylin-O-phenyl-monothiocarbonate (3, 0.53 g, 0.66 mmol, 87%) as a yellowish foam.

[0123] R f (n-Hex:EtOAc=2:1)=0.75。

[0124] 1 H NMR (CDCl3, 400 MHz): δ = 7.46-7.25 (m, 23H), 7.10-7.08 (m, 2H), 6.96 (d, J = 1.00, 8.66 Hz, 1H), 6.89 (s, 1H), 6.82 (s, 1H), 6.71 (d, J = 8.66 Hz, 1H), 5.36 (dd, J = 1.76, 12.92 Hz, 1H), 5.15 (s, 2H), 5.13 (s, 2H), 5.09 (m, 2H), 4.60 (s, 1H), 3.75-3.70 (m, 2H), 3.59 (d, J = 16.94 Hz, 1H) ppm。

[0125] 13 C NMR (CDCl3, 100 MHz): δ = 192.2, 153.1, 151.7, 149.3, 148.9, 148.0, 137.8, 137.4, 137.4, 137.3, 137.3, 134.9, 130.5, 129.6, 128.7, 128.6, 128.6, 128.6, 128.2, 128.0, 128.0, 127.7, 127.5, 127.4, 126.7, 124.5, 122.1, 115.5, 112.2, 111.8, 108.2, 89.9, 75.3, 72.1, 71.7, 71.5, 64.5, 49.3, 39.2 ppm。 TIFF2025114591000007.tif13170TIFF2025114591000008.tif53170

[0126] Alkene 4: 6S,11R-tetrabenzyl-hematoxylin-O-phenyl-monothiocarbonate (3, 470 mg, 0.59 mmol) was dissolved in dry toluene (15.0 mL) at room temperature under an inert gas atmosphere. The reaction mixture was vigorously stirred and heated to 115 °C (oil bath temperature) for 3 h, after which TLC analysis indicated complete conversion of 3. The reaction mixture was then cooled to room temperature and concentrated in vacuo. The crude alkene 4 thus obtained was purified by flash column chromatography (silica, n-Hex: EtOAc = 95:5 to 70:30 gradient) to give 3,4,9,10-tetrakis(benzyloxy)-6,7-dihydroindeno[2,1-c]chromene (4, 308 mg, 0.48 mmol, 81%) as a colorless solid (turns reddish upon exposure to air).

[0127] R f (n-Hex: EtOAc = 3:1) = 0.50.

[0128] 1 HNMR (CDCl3, 400 MHz): δ = 7.52-7.25 (m, 22H), 7.14 (s, 1H), 6.61 (d, J = 8.28 Hz, 1H), 5.21 (s, 2H), 5.19 (s, 2H), 5.15 (s, 2H), 5.11 (s, 4H), 3.33 (s, 1H) ppm.

[0129] 13 C NMR (CDCl, 100 MHz): δ = 152.7, 148.2, 147.8, 147.4, 137.9, 137.7, 137.5, 137.4, 137.2, 137.2, 135.3, 134.3, 131.8, 128.8, 128.7, 128.6, 128.6, 128.3, 128.0, 128.0, 128.0, 127.9, 127.7, 127.6, 127.5, 117.8, 116.5, 112.4, 109.4, 106.6, 75.3, 72.7, 72.0, 71.2, 67.2, 37.5 ppm. TIFF2025114591000009.tif13170TIFF2025114591000010.tif42170

[0130] rac-Benzyl ether 5: Alkene 4 (200 mg, 0.31 mmol, 1.00 equiv.) was dissolved in dry THF (5.0 mL) under an inert gas atmosphere at room temperature and subsequently cooled to 0–4 °C. Borane tetrahydrofuran complex (0.62 mL, 1 m in THF, 0.62 mmol, 2.00 equiv.) was then slowly added with stirring. The resulting mixture was stirred at room temperature for 4 h, and TLC analysis indicated complete consumption of 4. Absolute ethanol (1.00 mL) was then slowly added (caution: H evolution), and stirring was continued for an additional 10 min. Subsequently, aqueous sodium hydroxide (3 m, 2.0 mL) and hydrogen peroxide (30 wt%, 0.8 mL) were added, and the reaction was vigorously stirred for an additional 90 min. After dilution with dichloromethane (75 mL) and water (50 mL), the layers were separated, and the aqueous phase was extracted with dichloromethane (50 mL, twice). The combined organic layers were washed with brine (50 mL), dried over NaSO, filtered, and concentrated in vacuo. Final purification of crude rac-benzyl ether 5 was achieved by flash column chromatography (silica, n-Hex: EtOAc gradient from 80:20 to 60:40) to afford rac-tetrabenzyl-hematoxylin (5, 194 mg, 0.29 mmol, 94%) as an orange foam.

[0131] R f (n-Hex: EtOAc = 2:3) = 0.60.

[0132] 1 H NMR (DMSO-D6, 400 MHz): δ = 7.46-7.27 (m, 20H), 7.11-7.08 (s, 2H), 6.93 (s, 1H), 6.74 (d, J = 8.78 Hz, 1H), 5.14-5-04 (m, 6H), 4.91 (dd, J = 1.13, 11.67 Hz, 2H), 3.97 (s, 1H), 3.90 (d, J = 11.29 Hz, 1H), 3.76 (d, J = 11.04 Hz, 1H), 2.90 (s, 2H) ppm.

[0133] 1 H NMR (CDCl, 400 MHz), 13 C NMR (CDCl3, 100 MHz): δ = data consistent with that recorded for compound 2. TIFF2025114591000011.tif13170TIFF2025114591000012.tif42170

[0134] rac-Hemtoxylin 6 [1-3] rac-Tetrabenzyl-hematoxylin (5, 600 mg, 0.91 mmol, 1.00 equiv.) was dissolved in dry, degassed ethyl acetate (5.0 mL) at room temperature under an inert atmosphere. To this was added palladium on charcoal (96.3 mg, 10 wt.% Pd, 0.09 mmol, 0.10 equiv. Pd), and the atmosphere was replaced with hydrogen by evacuation and backfilling (three cycles). The resulting mixture was stirred vigorously for 2 days, after which TLC analysis showed complete conversion to product 6. The suspension was then filtered through a plug of degassed Celite (degassed ethyl acetate washes) and concentrated in vacuo under an inert atmosphere. The resulting orange glassy solid (250 mg) was crystallized from degassed water to give rac-hematoxylin (6, 187 mg, 0.62 mmol, 68%) as a yellowish-brown solid.

[0135] R f (n-Hex: EtOAc = 2:3) = 0.10.

[0136] 1 H NMR (DMSO-D6, 400 MHz): δ = 8.57 (s, br, 3H), 8.21 (s, br, 1H), 6.64-6.61 (m, 2H), 6.53 (s, 1H), 6.42 (d, J = 9.54 Hz, 1H), 5.27 (s, br, 1H), 3.92-3.86 (m, 2H), 3.59 (d, J = 10.29 Hz, 1H), 2.88 (d, J = 15.43 Hz, 1H), 2.72 (d, J = 15.31 Hz, 1H) ppm.

[0137] 13 C NMR (DMSO-D6, 100 MHz): δ = 144.2, 144.0, 143.9, 142.5, 135.6, 133.2, 129.7, 119.3, 115.2, 112.0, 111.7, 108.7, 76.3, 69.7, 49.9, 42.0 ppm. TIFF2025114591000013.tif13170

[0138] Example 3 - Equivalence analysis of synthetic hematoxylin Currently, hematoxylin is extracted from the heartwood of log trees by a few commercial producers. Often, the product varies from batch to batch and manufacturer to manufacturer, both in the level of impurities and in the ratio of hematoxylin to hematein. These factors suggest that synthetic hematoxylin could offer a more consistent and potentially superior alternative to the natural product currently offered by commercial producers.

[0139] The staining performance of synthetic hematoxylin was evaluated against commercially available hematoxylin (Lot #7740) purchased from Abbey Color, Inc. Racemic synthetic hematoxylin was produced according to the procedure described by Otto Dann and Hans Hofman, "Die Synthese von (+ / -)- Hamatoxylin, Chromane (XVII)," 1965, pages 1498-1504. Staining performance was evaluated across the suite of Ventana products containing hematoxylin. Stains included elastic NF hematoxylin, HE600 hematoxylin, trichrome hematoxylin, mucicarmine-iron hematoxylin, hematoxylin I, and hematoxylin II. For each stain evaluated, formulations were prepared using synthetic and commercially available hematoxylin. Comparability was determined using 16 paired analyses (32 slides each). All study slides were analyzed by a qualified reader.

[0140] In general, the performance of synthetic hematoxylin was comparable to or better than the natural product. All slides using synthetic preparations of Hem II were excellent, with nuclear chromatin detail often better than in preparations using the natural product. All special stains for which synthetic preparations were tested were comparable to or better than the natural preparations.

[0141] Materials and Methods Elastic NF hematoxylin was prepared according to Ventana MF 09186212001, rev C. HE600 hematoxylin was prepared according to Ventana MF 07283270001, rev M. Trichrome hematoxylin A was prepared according to Ventana MF 06518168001, rev F. Iron hematoxylin A was prepared according to Ventana MF 75001, rev P. In each case, 20 ml of each preparation was produced, sufficient for staining 100 slides each.

[0142] Preparation of Hematoxylin I: 0.3 g

[0143] material : Deionized water Ethylene Glycol, Part #10547 Hematoxylin dye, material #06002960001 or synthetic material Sodium iodate, BeanTown Chemical Co., catalog #212330-100G Aluminum Sulfate, Part #10357 Glacial Acetic Acid, VWR, Product #0714-500ML Tween 20, Sigma, Catalog #P-7949

[0144] procedure : Mix 56.25 ml of water with 20.87 g of ethylene glycol Add 0.30 g of hematoxylin and 36.0 mg of sodium iodate Stir at room temperature for 20 minutes Add 5.28g of aluminum sulfate and 1.5ml of acetic acid Stir at room temperature for 1 hour Filter the solution Add 75.0 mg of Tween 20

[0145] Preparation of Hematoxylin II: 0.15 g material : Deionized water Ethylene Glycol, Part #10547 Hematoxylin dye, material #06002960001 or synthetic material Sodium iodate, BeanTown Chemical Co., catalog #212330-100G Aluminum Sulfate, Part #10357 Glacial Acetic Acid, VWR, Product #0714-500ML Tween 20, Sigma, Catalog #P-7949

[0146] procedure : Mix 56.5 ml of water with 0.87 g of ethylene glycol Add 0.15 g of hematoxylin and 18.0 mg of sodium iodate Stir at room temperature for 20 minutes Add 1.32g of aluminum sulfate and 1.5ml of acetic acid Stir at room temperature for 1 hour Filter the solution Add 75.0 mg of Tween 20 NOTE: The solution should be stored at room temperature and allowed to age for 48 h before use.

[0147] Preparation of Hematoxylin I Dilution and Staining

[0148] (1) 72 mL of acetic acid / PEG200 stock was made using 11.6 mL of acetic acid (P / N 10040) and 62.4 mL of PEG200 (P / N 10068). Mix by inverting 10 times.

[0149] (2) Prepare a series of dilutions with the amounts of materials required listed below: Add the appropriate amount of water to a series of appropriately sized containers. Add PEG / acetic acid stock solution. Add the appropriate amount of hematoxylin stock. Mix by inverting 10 times. The dilutions are placed in labeled customer fillable dispensers and labeled with a generic counterstain barcode label.

[0150] (3) Test the dilutions using a certified Symphony MTB (P / N78T7000) using the following test protocol: Select the negative control mouse (Ig) P / N760-2014 as the primary antibody for the protocol. Select a 16 minute primary antibody incubation. For all hematoxylin dilutions, select a 4 minute counterstain incubation. Select Bluing Reagent (P / N 760-2037) incubation for 4 minutes. TIFF2025114591000014.tif98170

[0151] Preparation and staining of hematoxylin II dilutions

[0152] 1. Prepare a series of dilutions with the amounts of ingredients required, as listed below. Add the appropriate amount of water to a series of appropriately sized containers. Add the required amount of glacial acetic acid (P / N 10040). Add the required amount of polyethylene glycol 200 (P / N 10068). Add the required amount of in-house Hematoxylin II counterstain. Mix by inverting 10 times. Place the following dilutions into labeled customer fillable dispensers and label with a generic counterstain barcode label.

[0153] 2. Using a certified Symphony MTB (P / N78T7000), test the dilutions using the following test protocol: Select Negative Control Mouse (Ig)P / N760-2014 as the primary antibody in the protocol. Select a 16 minute primary antibody incubation. For all Hematoxylin II dilutions, select a 4 minute counterstain incubation. Select Bluing Reagent (P / N 760-2037) incubation for 4 minutes. TIFF2025114591000015.tif103170

[0154] After the procedure was completed, the slides were washed, dehydrated, and coverslipped according to OP2100-028 and OP2100-030.

[0155] The slides were read on a qualified reader and a titer of 58% at an incubation time of 4 minutes was selected as the standard formulation. TIFF2025114591000016.tif48170

[0156] conclusion Overall, the performance of the synthetic hematoxylins was comparable to or better than the natural preparations. The only situation in which synthetic performance was problematic was with the Hem I preparation, where the synthetic preparation appeared to give exaggerated cytoplasmic staining that was nearly as intense as the target nuclear staining. This appears to be a problem with the formulation, not the dye itself.

[0157] As shown in Figures 10-15, slides using synthetic HE600 preparations were superior to those using native preparations. All HE600 slides using synthetic preparations appeared clearer and provided more nuclear chromatin detail. All slides using synthetic Hem II preparations were excellent, often with better nuclear chromatin detail than native preparations. This is particularly evident in Figures 7A and 7B (colon) and 8A and 8B (kidney). All special stains tested with synthetic preparations were comparable to or better than native. In Figures 5A and 5B, staining with synthetic trichrome hematoxylin yielded comparable or better staining than native preparations. In the case of mucicarmine-iron-hematoxylin in Figures 6A and 6B, the synthetic preparation produced clearly superior staining with much higher contrast than the stain produced by the native preparation. It should be noted that although the synthetic hematoxylin produced an appropriate staining pattern on the elastic slides, the staining was significantly lighter than that of the commercial hematoxylin, which was expected since the synthetic hematoxylin was not completely soluble in ethanol when following the SOP for the elastic NF hematoxylin.

[0158] All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications referred to herein and / or listed in the Application Data Sheet are hereby incorporated by reference in their entirety. Aspects of the embodiments can be modified, if desired, to employ concepts from various patents, applications, and publications to provide further embodiments.

[0159] While the present disclosure has been described with reference to a number of exemplary embodiments, it should be understood that numerous other modifications and embodiments may be devised by those skilled in the art which fall within the spirit and scope of the principles of the present disclosure. More particularly, reasonable variations and modifications may be made in the component parts and / or arrangements of the combined configuration of the subject matter within the scope of the foregoing disclosure, the drawings, and the appended claims without departing from the spirit of the present disclosure. In addition to variations and modifications in the component parts and / or arrangements, alternative uses will also be apparent to those skilled in the art.

[0160] References: [1] Ch. Bettinger, HW Zimmermann, Hisotchemistry 1991, 96, 215-228. [2] JA Kiernan, Biotechnic & Histochemistry 2018, 93(2), 133-148. [3] O. Kel, A. Fuerstenberg, N. Mehanna, C. Niclas, B. Laleu, M. Hammarson, B. Albinsson, J. Lacour, E. Vauthey, Chem. Eur. J. 2013, 19, 7173-7180. [4] R. Corradini, S. Sforza, T. Tedeschi, R. Marchelli, Chirality 2007, 19, 269 -294. [5] Y. Xu, YX Zhang, H. Sugiyama, T. Umano, H. Osuga, K. Tanaka, J. Am. Chem. Soc. 2004, 126, 6566-6567. [6] HC Becker, B. Norden, J. Am. Chem. Soc.2000, 122, 8344-8349. [7] a) K. Okamoto, M. Noji, T. Tashiro, Y. Kidani, Chem. Pharm. Bull. 1981, 29, 929-939;b) K. Vickery, AM Bonin, RR Fenton, S. Omara, MJ McKeage, PJ Russell, TW Hambley, J. Med. Chem. 1993, 36, 3663-3668;c) RR Fenton, WJ Easdale, HM Er, SM Omara, MJ McKeage, PJ Russell, TW Hambley, J. Med.Chem.1997, 40, 1090-1098;e) J. Malina, J. Kas, V. Nabeckova, G. Nabeckova. Chem. Biol. 2002, 9, 629-638. [8] a) B. Norden, F. Tjerneld, FEBS Lett.1976, 67, 368-370;b) JK Barton, LA Basile, A. Danishefsky, A. Alexandrescu, Proc. Natl. Acad. Sci. USA 1984, 81, 1961-1965;c) A. Sitlani, EC Long, AM Pyle, JK Barton, J. Am. Chem.Soc.1992, 114, 2303-2312; e) CL Kielkopf, KE Erkkila, BP Hudson, JK Barton, DC Rees, Nat. Struct. Biol. 2000, 7, 117-121. [9] DL Boger, DS Johnson, Angew. Chem. They. Wheat. 1996, 35, 1438-1474.

[10] Wang AHJ, Ughetto G, Quigley GJ, Rich A, Biochemistry 1987, 26, 1152–1163.

[11] X. Qu, JO Trent, I Fokt, W Priebe, JB Chairs JB., Proc Natl Acad Sci USA 2000, 97, 12032-12037.

[12] a) MP Singh, B Plouvier, GC Hill, J Gueck, RT Pon, JW Lown, J Am. Chem. Soc. 1994, 116, 7006-7020;b) JWTrauger, Baird EE, Dervan PB, Nature 1996, 382, 559 -561; c) Kielkopf CL, White S, JW Szwewczyk, Turner JM, Baird EE, Dervan PB, Rees DC, Science 1998, 282, 111-115; (d) Herman DM, Baird EE, Dervan PB, J Am. Chem. Soc. Rev. 1998, 120, 1382–1391; e) PB Dervan, Bioorg. Med. Chem. Rev. 2001, 9, 2215–2235; f) PM Reddy, JW Toprowski, AL Kahane, TC Bruice, Bioorg.Med.Chem. Lett. Rev. 2005, 15, 5531–5536.

Claims

1. A hematoxylin preparation comprising a solvent, racemic hematoxylin, a chemical oxidizing agent, a mordant, a stabilizer, and an antioxidant.

2. 10. The hematoxylin preparation of claim 1, wherein the amount of chemical oxidizing agent present in the hematoxylin preparation is sufficient to convert at least a portion of the racemic hematoxylin to hematein.

3. 3. The hematoxylin preparation according to claim 1, wherein the solvent is an aqueous solvent and the antioxidant is a water-soluble antioxidant.

4. 4. The hematoxylin preparation of claim 3, wherein the water-soluble antioxidant comprises hydroquinone.

5. 10. The hematoxylin formulation of claim 1, wherein the solvent comprises one or more of water, a lower alkanol, and a polyol.

6. 10. The hematoxylin formulation of claim 1, wherein the solvent comprises water and a polyol.

7. 7. The hematoxylin formulation of claim 6, wherein the polyol is selected from the group consisting of propylene glycol, poly(ethylene glycol), and poly(propylene glycol).

8. 8. The hematoxylin preparation of claim 1, wherein between about 1% and about 50% of the hematoxylin is oxidized to hematein by the chemical oxidizing agent.

9. 9. The hematoxylin preparation of claim 1, wherein the chemical oxidizing agent is selected from the group consisting of sodium iodate, mercuric oxide, potassium permanganate, potassium periodate, hydrogen peroxide, and combinations thereof.

10. 9. The hematoxylin preparation of claim 1, wherein the chemical oxidizing agent comprises sodium iodate.

11. 11. The hematoxylin preparation of claim 1, wherein the mordant is selected from the group consisting of an aluminum mordant, an iron mordant, a bismuth mordant, a copper mordant, a molybdenum mordant, a vanadium mordant, a zirconium mordant, and combinations thereof.

12. 11. The hematoxylin preparation of claim 1, wherein the mordant comprises aluminum sulfate.

13. 13. The hematoxylin formulation of claim 1, wherein the stabilizer comprises a cyclodextrin or a cyclodextrin derivative.

14. 14. The hematoxylin formulation of claim 13, wherein the cyclodextrin or cyclodextrin derivative comprises β-cyclodextrin and β-cyclodextrin derivatives.

15. 15. The hematoxylin formulation of claim 1, further comprising an acid.

16. 16. The hematoxylin formulation of claim 1, wherein the hematoxylin formulation does not contain an acid additive.

17. 17. The hematoxylin formulation of claim 1, wherein the molar ratio of hematoxylin to oxidizing agent ranges between about 6:1 and about 1:

1.

18. 1. A method for staining a biological sample, comprising contacting the biological sample with a racemic hematoxylin preparation, the racemic hematoxylin preparation comprising a solvent, hematoxylin, a mordant, an antioxidant, a stabilizer, and a chemical oxidizing agent in an amount sufficient to convert at least a portion of the racemic hematoxylin to hematein.

19. 19. The method of claim 18, wherein the stabilizer is selected from the group consisting of polysaccharides, cryptands, cryptophanes, cavitands, crown ethers, dendrimers, nanotubes, calixarenes, valinomycin, nigericin, and combinations thereof.

20. 20. The method of claim 18 or 19, further comprising contacting the sample with a counterstain.

21. 21. The method of claim 20, wherein the counterstain is selected from the group consisting of Eosin Y, Orange G, Light Green SF Yellow, Bismarck Brown, and Fast Green FCF.

22. 22. The method of any one of claims 18 to 21, wherein contacting the sample with a hematoxylin preparation comprises a progressive hematoxylin staining protocol.

23. 22. The method of any one of claims 18 to 21, wherein contacting the sample with a hematoxylin preparation comprises a recurrent hematoxylin staining protocol.

24. 24. The method of any one of claims 18 to 23, wherein the method is automated.

25. 25. The method of any one of claims 18 to 24, wherein the biological sample is supported on a substrate.

26. 26. The method of claim 25, wherein the substrate comprises a microscope slide.

27. 27. The method of any one of claims 18 to 26, wherein the biological sample comprises a histological or cytological sample.

28. 28. The method of claim 27, wherein the method is automated.

29. 29. The method of any one of claims 18 to 28, wherein the stabilizer has an aqueous solubility of greater than about 100 mg / mL at about 25°C.

30. 30. The method of any one of claims 18 to 29, wherein the antioxidant is selected from the group consisting of hydroquinone, n-alkyl gallates, reducing sugars, benzoates, hydroxybenzoates, sulfites, metabisulfites, citric acid, tartaric acid, lactic acid, erythorbic acid, ascorbic acid, uric acid, tannic acid, chelating agents, coral hydrate, and derivatives and salts thereof.

31. 30. The method according to any one of claims 18 to 29, wherein the racemic hematoxylin preparation is a hematoxylin preparation according to any one of claims 1 to 17.

32. 1. A pH-reduced hematoxylin formulation comprising racemic hematoxylin and an acid in an amount ranging from about 0.2% to about 4% of the total volume of the pH-reduced hematoxylin formulation, wherein the pH-reduced hematoxylin formulation has a pH value of less than about 2.4, and the acid is selected from the group consisting of hydrochloric acid, sulfuric acid, perchloric acid, and nitric acid.

33. 33. The reduced pH hematoxylin formulation of claim 32, wherein the pH value is less than about 2.

3.

34. 33. The reduced pH hematoxylin formulation of claim 32, wherein the pH value is less than about 2.

2.

35. 33. The reduced pH hematoxylin formulation of claim 32, wherein the pH value is less than about 2.

1.

36. 33. The reduced pH hematoxylin formulation of claim 32, wherein the pH value is in the range between about 2.1 and about 2.

2.

37. 37. The reduced pH hematoxylin formulation of any one of claims 32 to 36, wherein the acid is selected from the group consisting of hydrochloric acid, sulfuric acid, perchloric acid, and nitric acid.

38. 38. The reduced pH hematoxylin formulation of any one of claims 32 to 37, further comprising a mordant and an oxidizing agent.

39. 39. The reduced pH hematoxylin formulation of any one of claims 32 to 38, further comprising a polyol.

40. 40. The reduced pH hematoxylin formulation of claim 39, wherein the polyol is selected from the group consisting of propylene glycol, ethylene glycol, and mixtures thereof.

41. 33. The reduced pH hematoxylin formulation of claim 32, wherein the formulation consists essentially of racemic hematoxylin, a mordant, an oxidizing agent, and an acid.

42. 39. The reduced pH hematoxylin of claim 38, wherein the oxidizing agent comprises sodium iodate and the mordant comprises aluminum.

43. 43. A method of staining a biological sample, the method comprising: increasing the pH value of an aliquot of the pH-reduced hematoxylin formulation of any one of claims 32 to 42 to provide a pH-adjusted racemic hematoxylin formulation solution having a pH value in the range of about 2.4 to about 2.6; and contacting the biological sample with the pH-adjusted racemic hematoxylin formulation having a pH value in the range of about 2.4 to about 2.

6.

44. 44. The method of claim 43, wherein the pH value of the aliquot of the pH-reduced hematoxylin preparation is increased by adding a readjustment solution comprising a strong base and / or a buffer.

45. 45. The method of claim 44, wherein the strong base is selected from the group consisting of sodium hydroxide, potassium hydroxide, lithium hydroxide, and ammonia.

46. The weak acid of the buffer has a pK in the range of about 1.5 to about 3.

5. ａ 45. The method of claim 44, comprising:

47. 45. The method of claim 44, wherein the reconditioning solution further comprises an additive selected from the group consisting of a polyol, an oxidizing agent, a mordant, and any combination thereof.

48. 48. The method of any one of claims 43 to 47, wherein the pH value of the pH-reduced hematoxylin preparation is increased before contacting the biological sample with the pH-adjusted racemic hematoxylin preparation.

49. 49. The method of any one of claims 43 to 48, wherein the biological sample comprises a histological or cytological sample.

50. A kit comprising a first component and a second component, the first component comprises a pH-reduced hematoxylin formulation comprising racemic hematoxylin and an acid in an amount ranging from about 0.1% to about 10% of the total volume of the pH-reduced hematoxylin formulation, the pH-reduced hematoxylin formulation having a pH value of less than about 2.4; and the second component comprises a strong base or buffer and is provided in an amount relative to the first component such that when the first and second components are mixed, the pH value of the pH-reduced hematoxylin formulation is raised to above about 2.4; kit.

51. 51. The kit of claim 50, wherein the second component is provided in an amount relative to the first component such that, when the first and second components are mixed, the pH value of the pH-reduced hematoxylin formulation is raised to above about 2.

5.

52. 51. The kit of claim 50, wherein the second component is provided in an amount relative to the first component such that, when the first and second components are mixed, the pH value of the pH-adjusted racemic hematoxylin formulation is in the range of between about 2.45 and about 2.

54.

53. 51. The kit of claim 50, wherein the second component is provided in an amount relative to the first component such that, when the first and second components are mixed, the pH-adjusted racemic hematoxylin formulation has a pH value in the range of between about 2.5 and about 2.

6.

54. 54. The kit of any one of claims 50 to 53, wherein the first component further comprises a mordant and an oxidizing agent.

55. 55. The kit of claim 54, wherein the first component further comprises a shelf-life extender.

56. 56. The kit of claim 55, wherein the shelf-life extending agent comprises a polyol.

57. 57. The kit of claim 56, wherein the polyol is selected from the group consisting of propylene glycol, ethylene glycol, and mixtures thereof.

58. 51. The kit of claim 50, wherein the reduced pH hematoxylin formulation consists essentially of racemic hematoxylin, a mordant, an oxidizing agent, and an acid.

59. Reduced pH hematoxylin 59. A kit according to any one of claims 50 to 58, wherein the formulation is a reduced pH hematoxylin formulation according to any one of claims 32 to 42.

60. 60. The kit of any one of claims 50 to 59, wherein the second component comprises a strong base selected from the group consisting of sodium hydroxide, potassium hydroxide, and lithium hydroxide.

61. 60. The kit of any one of claims 50 to 59, wherein the second component comprises a buffer selected from the group consisting of cyanoacetate, fumarate, and sulfate.

62. 62. The kit of any one of claims 50 to 61, further comprising a third component comprising a counterstain.

63. 63. The kit of claim 62, wherein the counterstain is selected from the group consisting of Eosin Y, Orange G, Light Green SF Yellow, Bismarck Brown, and Fast Green FCF.

64. 1. A system for staining a biological sample mounted on a substrate, comprising: a first container containing a pH-reduced hematoxylin formulation comprising racemic hematoxylin and an acid in an amount ranging from about 0.1% to about 10% of the total volume of the pH-reduced hematoxylin formulation, wherein the pH-reduced hematoxylin formulation has a pH value of less than about 2.4; a second container containing a reconditioning solution, the first container and the second container being fluidly connected to the mixing container such that the pH-reduced hematoxylin formulation and the reconditioning solution can be combined to provide the hematoxylin staining solution; Including, the system.

65. 65. The system of claim 64, further comprising a substrate holder for holding a biological sample mounted on a substrate, the substrate holder being in fluid communication with the mixing vessel so that the hematoxylin staining solution can be applied to the biological sample.

66. 66. The system of claim 64 or 65, wherein the pH-reduced hematoxylin formulation is a pH-reduced hematoxylin formulation described in any one of claims 32 to 42.

67. Use of racemic hematoxylin for staining biological specimens.

68. 68. The use according to claim 67, wherein the biological sample is a histological sample.

69. 68. The use according to claim 67, wherein the biological sample is a cytological sample.

70. 1. Use of a hematoxylin preparation for staining a biological sample, the hematoxylin preparation comprising a solvent, racemic hematoxylin, a chemical oxidizing agent, a mordant, a stabilizer, and an antioxidant.

71. 71. The use according to claim 70, wherein the biological sample is a histological sample.

72. 71. The use according to claim 70, wherein the biological sample is a cytological sample.

73. 73. The use according to any one of claims 70 to 72, wherein the staining of the biological sample is automated.

74. 74. Use according to any one of claims 70 to 73, wherein the hematoxylin preparation is a hematoxylin preparation according to any one of claims 1 to 17.

75. A hematoxylin staining composition comprising a solvent, racemic hematoxylin, a chemical oxidizing agent in an amount sufficient to convert at least a portion of the racemic hematoxylin to hematein, and a mordant, wherein the composition contains chloride (Cl － ) and sulfate (SO ４ ２－ ), wherein the chloride / sulfate molar ratio ranges between about 2.5:1 and about 1:

4.

76. 76. The hematoxylin staining composition of claim 75, wherein the chloride / sulfate molar ratio ranges between about 2:1 and about 1:

2.

77. 76. The hematoxylin staining composition of claim 75, wherein the chloride / sulfate molar ratio is in the range of between about 1.5:1 and about 1:1.

5.

78. 76. The hematoxylin staining composition of claim 75, wherein the chloride / sulfate molar ratio ranges between about 1:1 and about 1:

1.

79. 79. A hematoxylin staining composition according to any one of claims 75 to 78, wherein the racemic hematoxylin has a molar concentration ranging from about 0.01 M to about 0.05 M.

80. 1. A method for producing a hematoxylin formulation, comprising: adding hematoxylin to a solvent; adding a chemical oxidizing agent in an amount sufficient to convert at least a portion of the hematoxylin to hematein; and adding a mordant and a counterion, wherein the formulation has a chloride / sulfate molar ratio, and the method further comprises the step of modifying the chloride / sulfate molar ratio to a molar ratio in the range of between about 2.5:1 and about 1:4.

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