Materials and methods for bleaching melanin-pigmented tissue

Abstract

A method for depigmenting melanin pigmented samples is provided. The samples are incubated in the presence of a hydrogen peroxide-based solution at a temperature below 65° C. for up to 180 minutes. Time, temperature and hydrogen peroxide concentrations that appropriately balance the degree of depigmentation with the maintenance of cell morphology and sample retention are also disclosed.

Description

[Technical field]

[0001] background I. Field of the Invention

[0002] The disclosed invention relates to materials and methods useful in the field of preparation of cell samples for microscopic analysis, particularly pigmented tissue. [Background technology]

[0003] II. Brief Description of Related Art

[0004] Melanin is a naturally occurring, stable, insoluble pigment present in neoplastic and non-neoplastic tissues that can interfere with histopathological evaluation of pigmented lesions by obscuring cellular morphology. Furthermore, chromogens used in immunohistochemistry (IHC) analysis can be masked by brown-black pigments, complicating IHC biomarker evaluation and therefore patient diagnosis.

[0005] A variety of manual and semi-automated protocols to mitigate these issues have been reviewed in the literature.

[0006] Chung et al. used 0.5% hydrogen peroxide (H 2 O 2 We describe a manual protocol for removing excess melanin from 5 μm-thick heavily pigmented formalin-fixed paraffin-embedded (FFPE) melanoma tissues using a 0.5% H2O2 filter with incubation at a relatively high temperature, followed by immunohistochemistry (IHC) staining for various biomarkers. The results were visualized and tissue morphology was preserved. 2 O 2 The solution was used (80°C).

[0007] Liu et al. described the process of an automated immunohistochemical staining platform that uses hydrogen peroxide diluted in phosphate-buffered saline at 65 °C to depigment melanin in 3-μm-thick ocular and cutaneous melanoma samples.

[0008] Manicam et al. evaluated the effect of different bleaching reagents and conditions on melanin removal. The study was limited to hematoxylin and eosin (H&E) staining after bleaching and did not test IHC staining.

[0009] Momose et al. 2 O 2 The bleaching performance of the 100% 100% 100% 15 ... 2 O 2 This was done manually using the solution.

[0010] Orchard et al. (2019) described a semi-automated bleaching procedure that tested a variety of biomarkers, including Mart1, S-100, Sox-10, HMB45, and CD68. However, the bleaching procedure was performed off-line from the staining system. Furthermore, very high concentrations of H 2 O 2 (10% final concentration in PBS) was used.

[0011] Foss et al. investigated the effect of bleaching using potassium permanganate on antigen and IHC staining procedures (i.e., pre- and post-antigen retrieval, post-antibody introduction, post-detection chemistry). They found that the order of bleaching before or after antigen retrieval made no difference to antigenicity but significantly affected tissue loss. Little tissue loss was observed when antigen retrieval was performed after bleaching. It was also demonstrated that the detection chemistry was sensitive to oxidation, resulting in decolorized reaction products.

[0012] Orchard (2007) investigated the effect of temperature on the efficiency of melanin bleaching. After bleaching, various biomarkers such as S100, HMB45, NKIC3, Melan-A, CD3, CD20, CD68, CD34, CD45, CD31, and SMA were used. Melanin bleaching was demonstrated to be more efficient at higher temperatures (i.e., 60°C) than at temperatures close to room temperature (i.e., 37°C).

[0013] applied different bleaching formulations to melanotic malignant melanomas before and after an immunoperoxidase staining sequence. The tissues were stained for S100 and NSE biomarkers. Pretreatment with potassium permanganate and oxalic acid was found to be the optimal bleaching method.

[0014] Hu et al. used Sox-10, S-100, HMB45 and Melan-A in IHC staining under various bleaching conditions (i.e., H 2 O 2 They tested 30% H for 24 hours at 24°C. 2 O 2 The optimized bleaching conditions were found.

[0015] To the best of our knowledge, a fully automated protocol that adequately balances adequate depigmentation, flexibility in antigen retrieval, preservation of morphology, and mitigation of tissue loss has not yet been developed. Summary of the Invention

[0016] The present disclosure relates to methods, reagents, and devices for removing melanin pigmentation from cell samples using a hydrogen peroxide solution. The disclosed methods are fully automatable, yet result in samples that are sufficiently depigmented to allow IHC analysis, while maintaining proper cell morphology and limiting sample loss. The disclosed methods generally involve removing cell samples from a buffered H 2 O 2and incubating in aqueous solution at a concentration of up to 5% for up to 180 minutes at a temperature of less than 65° C. Also disclosed are automated methods of affinity staining cell samples incorporating the disclosed methods, as well as devices and reagents for carrying out the same. [Brief description of the drawings]

[0017] [Figure 1] FIG. 1 shows an exemplary automated workflow for depigmenting melanin pigment samples with an automated affinity stainer. [Diagram 2] 1 is a series of images illustrating the automated depigmentation method. [Figure 3A] This is a Pareto chart derived from Table 3. [Figure 3B] Figure 1 shows the main effects derived from Table 3. [Figure 3C] 13 is a plot of interactions derived from Table 3. [Figure 4A] This is a Pareto chart derived from Table 5. [Figure 4B] Figure 1 shows the main effects derived from Table 5. [Figure 4C] 13 is a plot of interactions derived from Table 5. [Diagram 5] A representative series of images of concentration and time optimization at 37° C. and 50° C. [Figure 6] 13 is a series of representative images from evaluation of long incubation times. [Figure 7] 1 is a series of representative images of an evaluation of diluent composition and paddle conditions. [Figure 8] 1 shows the effect of various Tris concentrations in paddle buffer on destaining performance. [Figure 9] 1 shows a stability study of a 10% H2O2 stock solution based on pH change over time in various diluents. [Figure 10] 1 shows a stability study of a 10% H2O2 stock solution based on changes in H2O2 concentration over time in various diluents. [Figure 11A]Melanin intensity for hypermelanin cases using stock solutions of 1-20% H2O2 in water (working concentrations of 0.25%-5% in 75 mM Tris) for 92 min at 37 °C are shown. [Figure 11B] Melanin coverage of hypermelanotic cases using stock solutions of 1-20% H2O2 in water (working concentrations of 0.25%-5% in 75 mM Tris) for 92 min at 37 °C is shown. [Figure 11C] Representative images of samples depigmented for 92 min in a stock solution of 1–20% HO in water at 37 °C (working concentrations of 0.25%–5% in 75 mM Tris) are shown. [Figure 11D] Representative images of samples depigmented in a preservative solution of 1-20% HO in water (working concentration of 0.25%-5% in 75 mM Tris) for 92 min at 37 °C and immunohistochemically stained for LAG3 are shown. [Figure 12A] Representative images of samples depigmented in 10% H2O2 stock solution in water (2.5% working concentration in 75 mM Tris) for 92 min at room temperature (20°C), 37°C, 45°C, 55°C, 65°C or 75°C are shown. [Figure 12B] Melanin intensity for high melanin cases using 10% H2O2 stock solution in water (2.5% working concentration in 75 mM Tris) for 92 minutes at room temperature (20°C), 37°C, 45°C, 55°C, 65°C or 75°C is shown. [Figure 12C] Melanin coverage for high melanin cases using 10% H2O2 in water (2.5% working concentration in 75 mM Tris) for 92 minutes at room temperature (20°C), 37°C, 45°C, 55°C, 65°C or 75°C is shown. [Figure 13A] Melanin intensity for the hypermelanin case using a stock solution of 10% H2O2 in water (working concentration of 2.5% in 75 mM Tris) for 64, 72, 80, 92, or 180 minutes at 37°C is shown. [Figure 13B] Melanin coverage of hypermelanotic cases using a stock solution of 1-20% H2O2 in water (working concentration of 2.5% in 75 mM Tris) for 64, 72, 80, 92, or 180 min at 37 °C is shown. [Figure 13C]Representative images of samples depigmented in a stock solution of 1-20% HO in water (working concentration of 2.5% in 75 mM Tris) for 64, 72, 80, 92, or 180 min at 37 °C are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] Detailed Description of the Invention I. Abbreviations and Definitions

[0019] Unless otherwise defined, technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art.See, for example, Lackie, DICTIONARY OF CELL AND MOLECULAR BIOLOGY, Elsevier (4th ed.2007); Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, Cold Springs Harbor Press (Cold Springs Harbor, NY1989). The term "a" or "an" is intended to mean "one or more". The term "comprise", "comprises", "comprising", when preceding a list of steps or elements, is intended to mean that the addition of further steps or elements is optional and not excluded.

[0020] Affinity detection: A process that involves labeling a biomarker in a cell sample with a biomarker-specific reagent and a detection reagent in a manner that allows for microscopic detection of the biomarker. Examples include immunohistochemistry (IHC), chromogenic in situ hybridization (CISH), fluorescent in situ hybridization (FISH), silver in situ hybridization (SISH); and staining of formalin-fixed, paraffin-embedded tissue sections.

[0021] Antibody: The term "antibody" as used herein is used in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies, (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0022] Antibody fragment: "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0023] Biomarker: As used herein, the term "biomarker" refers to any molecule or group of molecules found in a biological sample that can be used to characterize the biological sample or the subject from which the biological sample is obtained. For example, a biomarker can be a molecule or group of molecules whose presence, absence, or relative abundance is characteristic of a particular cell or tissue type or condition; a particular pathological condition or condition; or the severity of a pathological condition, the likelihood of progression or regression of a pathological condition, and / or the likelihood that a pathological condition will respond to a particular treatment. As another example, a biomarker can be a cell type or microorganism (such as bacteria, mycobacteria, fungi, viruses, etc.), or a substitute molecule or group of molecules thereof.

[0024] Biomarker-specific reagent: A specific detection reagent, such as a primary antibody, that can bind directly and specifically to one or more biomarkers in a cell sample.

[0025] Cell sample: As used herein, the term "cell sample" refers to any sample containing intact cells, such as a cell culture, a body fluid sample, or a surgical specimen taken for pathological, histological, or cytological interpretation.

[0026] DAB: 3,3'-diaminobenzidine.

[0027] Detection Reagent: A "detection reagent" is any reagent used to deposit a detectable moiety near a biomarker-specific reagent in a cell sample. Non-limiting examples include biomarker-specific reagents (such as a primary antibody), secondary detection reagents (such as a secondary antibody that can bind to a primary antibody), tertiary detection reagents (such as a tertiary antibody that can bind to a secondary antibody), enzymes directly or indirectly associated with the biomarker-specific reagent, chemicals reactive with such enzymes that result in attachment of a detectable moiety (e.g., fluorescent or chromogenic dyes), washing reagents used between staining steps, and the like.

[0028] Detectable moiety: A molecule or material capable of producing a detectable signal (e.g., visual, electrical, or other) that indicates the presence and / or amount of a detectable moiety deposited in a sample. The detectable signal may be produced by any known or yet to be discovered mechanism, including absorption, emission, and / or scattering of photons (including radio frequency, microwave frequency, infrared frequency, visible frequency, and ultraviolet frequency photons). The term "detectable moiety" includes chromogenic, fluorescent, phosphorescent, and luminescent molecules and materials, catalysts (such as enzymes) that convert one substance to another to provide a detectable difference (by converting a colorless substance to a colored substance or vice versa, or by producing a precipitate or increasing the turbidity of the sample). In some examples, the detectable moiety is a fluorophore, which belongs to several common chemical classes, including coumarins, fluoresceins (or fluorescein derivatives and analogs), rhodamines, resorufins, luminophores, and cyanines. Additional examples of fluorescent molecules can be found in Molecular Probes Handbook-A Guide to Fluorescent Probes and Labeling Technologies, Molecular Probes, Eugene, OR, ThermoFisher Scientific, 11 thIn other embodiments, the detectable moiety is a molecule detectable by bright field microscopy, such as dyes including diaminobenzidine (DAB), 4-(dimethylamino)azobenzene-4'-sulfonamide (DABSYL), tetramethylrhodamine (DISCOVERY Purple), N,N'-biscarboxypentyl-5,5'-disulfonato-indo-dicarbocyanine (Cy5), and rhodamine 110 (Rhodamine).

[0029] Monoclonal antibody: An antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies constituting the population are identical and / or bind the same epitope, with the exception of variant antibodies that contain naturally occurring mutations or may arise during the manufacture of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" denotes the character of the antibody obtained from a substantially homogeneous population of antibodies, and is not constructed to require production of the antibody by any particular method. For example, the monoclonal antibodies used in accordance with the present invention may be made by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, or combinations thereof.

[0030] NRC: negative reagent control.

[0031] Sample: As used herein, the term "sample" is intended to refer to any material obtained from a subject that can be tested for the presence or absence of a biomarker.

[0032] Secondary detection reagent: A specific detection reagent capable of specifically binding to a biomarker-specific reagent.

[0033] Section: as a noun, a thin section of a tissue sample suitable for microscopic analysis, usually cut with a microtome; as a verb, the process of making sections.

[0034] Specific detection reagent: any composition of matter that can specifically bind to a chemical structure of a target in the context of a cell sample. As used herein, the phrases "specific binding," "specifically binds to," or "specific for" refer to a measurable and reproducible interaction between a target and a specific detection reagent that determines the presence of the target in the presence of a heterogeneous population of molecules, including biological molecules. For example, an antibody that specifically binds to a target is an antibody that binds to this target with higher affinity, avidity, more readily, and / or with a longer duration than it binds to other targets. In one embodiment, the extent to which a specific detection reagent binds to an unrelated target is less than about 10% of the binding of an antibody to the target, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, a biomarker-specific reagent that specifically binds to a target has a dissociation constant (Kd) of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, or ≦0.1 nM. In another embodiment, specific binding can include, but is not required to include, exclusive binding.Exemplary specific detection reagents include nucleic acid probes specific for particular nucleotide sequences, antibodies and antigen-binding fragments thereof, as well as antibodies and antigen-binding fragments thereof, such as ADNECTIN (10th FN3 fibronectin-based scaffold; Bristol-Myers-Squibb Co.), AFFIBODY (scaffold based on the Z domain of protein A from S. aureus; Affibody AB, Solna, Sweden), AVIMER (domain A / LDL receptor-based scaffold; Amgen, Thousand Oaks, Calif.), dAb (scaffold based on VH or VL antibody domains; GlaxoSmithKline PLC, Cambridge, UK), DARPIN (ankyrin repeat protein-based scaffold; Molecular Partners AG, Zurich, Switzerland), ANTICALIN (lipocalin-based scaffold; Pieris AG, Phillysing, Germany), NANOBODY (VHH (camelid Ig-based scaffold); Ablynx), and other antibody-specific detection reagents. N / V, Ghent, Belgium), TRANS-BODY (a transferrin-based scaffold; Pfizer Inc., New York, NY), SMIP (Emergent Biosolutions, Inc., Rockville, MD), and TETRANECTIN (a C-type lectin domain (CTLD)-based scaffold), tetranectin; Borean Pharma A / S, Aarhus, Denmark). A description of such engineered specific binding structures is reviewed in Wurch et al., Development of Novel Protein Scaffolds as Alternatives to Whole Antibodies for Imaging and Therapy: Status on Discovery Research and Clinical Validation, Current Pharmaceutical Biotechnology, Vol. 9, pp. 502-509 (2008), the contents of which are incorporated by reference.

[0035] Stain: When used as a noun, the term "stain" shall refer to any substance that can be used to visualize specific molecules or structures in a cell sample for microscopic analysis, including bright field microscopy, fluorescence microscopy, electron microscopy, etc. When used as a verb, the term "stain" shall refer to any process that results in the deposition of a colorant on a cell sample.

[0036] Subject: As used herein, the term "subject" or "individual" refers to a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs and horses), primates (e.g., humans and non-human primates, such as monkeys), rabbits and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

[0037] Test Sample: A tumor sample obtained from a subject whose outcome is unknown at the time the sample is obtained.

[0038] Tissue Sample: As used herein, the term "tissue sample" is intended to refer to a sample of cells that preserves the cross-sectional spatial relationships among cells that were present within the subject from which the sample was obtained.

[0039] Tumor sample: A tissue sample obtained from a tumor.

[0040] II. Methods of depigmentation

[0041] The inventors sought to identify a fully automatable methodology for removing melanin pigment from cell samples that would meet all of the following criteria: (A) reduce melanin pigment to a level that allows specific interpretation of DAB-stained samples; (B) use only reagent formulations that are storage stable, compatible with commercially available staining devices, and pose minimal environmental risks; (C) maintain acceptable morphology without unacceptable sample loss; (D) do not substantially interfere with subsequent affinity histochemical or cytochemical analysis. Although many depigmentation methodologies are known in the art, none were identified that met each of these criteria. The inventors have discovered that a hydrogen peroxide depigmentation method can be adapted for use in an automated affinity staining platform that reaches each of the aforementioned criteria.

[0042] A. Sample

[0043] The sample is a cell sample from melanin-pigmented tissue, such as a tissue sample (including a biopsy sample or tumor resection) or a cytological sample (such as a fine needle aspirate). In some embodiments, the cell sample is obtained from a subject having or suspected of having a tumor. In some embodiments, the sample is obtained directly from the tumor. In some embodiments, the tumor is a solid tumor, such as a carcinoma, lymphoma, or sarcoma. In embodiments, the cell sample is derived from a suspected or confirmed melanoma. When a tissue sample is used, the tissue sample is processed in a manner compatible with histochemical staining, including, for example, fixation, embedding in a wax matrix (such as paraffin), and sectioning (such as by a microtome). No specific processing steps are required by the present disclosure, so long as the sample obtained is compatible with affinity staining. In certain embodiments, microtome sections of formalin-fixed, paraffin-embedded (FFPE) samples are used in the staining process.

[0044] B. Bleaching reaction environment

[0045] The bleaching method is to bleach the cell sample by soaking it in a 1% to 5% aqueous solution of hydrogen peroxide (referred to herein as H 2 O 2 This is done by contacting the sample with a bleaching solution (called a bleaching solution) and incubating the sample for up to 180 minutes at a temperature below 65 °C. This combination of hydrogen peroxide concentration, temperature and time has been found to remove sufficient amounts of melanin from the sample to allow interpretation of the DAB staining without causing substantial sample loss or unacceptable morphological changes.

[0046] H 2 O 2 The bleaching solution should contain at least 1% and up to 5% (w / w) H 2 O 2 Including H 2 O 2 Exemplary ranges of concentrations include 1%-5%, 1.25%-5%, 2%-5%, 2.5%-5%, 2%-4%, 1.0%-3%, 1.0%-2%, 1.25%-2.5%, 2.0%-3%, 2.5%-3.5%, 3%-4%, 3.5%-4.5%, 4%-5%, 1.0%-1.5%, 1.5%-2.0%, 2.0%-2.5%, 2.5%-3.0%, 3.0%-3.5%, 3.5%-4.0%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, and about 5%.

[0047] H 2 O 2The bleaching solution may include a buffer to aid in achieving and maintaining a particular pH range. Exemplary buffers include citrate, tris(hydroxymethyl)aminomethane ("Tris"), phosphate buffers, and the like. Unless otherwise specified, the listed pH of a solution refers to the pH of the solution as measured at 20° C. The pH of the aqueous solution is generally greater than pH 4, preferably at least pH 7 (e.g., pH 7.4 or greater), and may even be as high as pH 9 or greater. Exemplary pH ranges expressly contemplated include pH 7-pH 9, pH 7.4-pH 9, pH 7-pH 8, and pH 8-pH 9. In an exemplary embodiment, the aqueous environment includes a Tris buffer in the range of 5 mM-250 mM and has a pH in the range of 7.0-9. In another exemplary embodiment, the aqueous environment includes 5 mM-75 mM Tris and a pH in the range of 7.0 to 8.0. In another exemplary embodiment, the aqueous environment comprises 75 mM to 250 mM Tris and has a pH in the range of 7.0 to 8.5. In another exemplary embodiment, the aqueous environment comprises 7.5 mM to 175 mM Tris and has a pH in the range of 7.9 to 8.8. In another exemplary embodiment, the aqueous environment comprises 0 mM to 170 mM Tris and has a pH in the range of 7.0 to 8.8. In another exemplary embodiment, the aqueous environment comprises 10 mM to 70 mM Tris and has a pH in the range of 7.7 to 8.6. In another exemplary embodiment, the aqueous environment comprises 20 mM to 80 mM Tris and has a pH in the range of 7.9 to 9.0. In another exemplary embodiment, the aqueous environment comprises 30 mM to 90 mM Tris and has a pH in the range of 8.0 to 8.7. In another exemplary embodiment, the aqueous environment comprises 50 mM to 110 mM Tris and has a pH in the range of 8.0 to 8.8. In another exemplary embodiment, the aqueous environment comprises 100 mM to 160 mM Tris and has a pH in the range of 8.2 to 8.9. In another exemplary embodiment, the aqueous environment comprises 150 mM to 200 mM Tris and has a pH in the range of 8.3 to 8.9.

[0048] Incubation is generally carried out at a temperature below 65° C. (preferably below 55° C., more preferably below 50° C.) for up to 180 minutes. The exact amount of time selected depends on the temperature of the H 2 O 2 The increase in the concentration of H depends on the concentration of H and the temperature of incubation, with higher concentrations and temperatures both allowing for shorter periods. Above 65°C and 180 min, 2 O 2 Bleaching solutions have an unacceptable tendency to cause morphological damage and loss of sample. At 55°C, H 2 O 2 Bleaching solutions generally result in acceptable morphology and bleaching, but still tend to cause tissue loss and some morphological damage. Below 50°C, H 2 O 2 Bleaching solutions generally provide acceptable morphology and bleaching with little tissue loss or morphological damage.

[0049] In an exemplary embodiment, the temperature is selected between 20° C. and 55° C. This temperature range generally allows acceptable destaining, allowing immunohistochemical analysis by DAB staining, without causing unacceptable morphological damage or sample loss. Within this range, lower temperatures require longer incubation times and result in less destaining, while higher temperatures require shorter incubations and tend to increase the degree of destaining, but at the same time at a higher risk of causing morphological damage and sample loss.

[0050] In another exemplary embodiment, the temperature is between 37° C. and 50° C. Within this temperature range, the degree of depigmentation (as measured by melanin intensity and melanin coverage) is improved compared to 20° C., and the incidence of morphological damage and sample loss is reduced compared to 55° C.

[0051] In another exemplary embodiment, the temperature is between 37° C. and 45° C. Within this temperature range, the degree of depigmentation (as measured by melanin intensity and melanin coverage) is improved compared to 20° C., but the degree of morphological damage and sample loss is further improved compared to 55° C.

[0052] In another embodiment, the temperature is about 37°C to about 45°C, and the time is 8 minutes to 180 minutes. In another embodiment, the temperature is about 37°C to about 45°C, and the time is 60 minutes to 180 minutes. 2 O 2 The bleach solution should be 2% to 4% H2O with a pH in the range of 7.0 to 8.5. 2 O 2 and up to 250 mM Tris, the temperature is within the range of about 37° C. to about 45° C., and the time is 60 minutes to 180 minutes. 2 O 2 The bleach solution should be 2% to 4% H2O with a pH in the range of 7.0 to 8.5. 2 O 2 and up to 250 mM Tris, the temperature is within the range of about 37° C. to about 45° C., and the time is 60 minutes to 95 minutes. 2 O 2 The bleach solution is approximately 2.5% H2SO4 with a pH in the range of 7.0 to 8.5. 2 O 2 and up to 250 mM Tris, at a temperature in the range of about 37° C. to about 45° C., for a time period of 60 minutes to 180 minutes. 2 O 2 The bleach solution is about 2.5% H2O at a pH of about 8. 2 O 2 and about 75 mM Tris, the temperature is in the range of 37° C. to 45° C., and the time is 60 minutes to 95 minutes.

[0053] In the context of pH, the term "about" shall be construed to mean a pH that, when rounded to the nearest hundredth (per NCES Standard 5-3), is within 5% of the recited pH. For example, a pH of "about 7" should include any pH within pH 7 ±0.35.

[0054] In the context of concentrations, the term "about" shall be interpreted to mean a concentration that is within 10% of the recited concentration when rounded to the nearest hundredth in the same units of measurement (per NCES Standard 5-3). For example, a concentration of "about 100 mM" shall include any concentration rounded to within 100 mM ± 10 mM. Similarly, a concentration of "about 7.5% w / w" shall be interpreted to mean 7.5% w / w ± 0.75%.

[0055] In the context of temperature, the term "about" shall be construed to mean a temperature that is within 5% of the recited temperature when rounded to the nearest hundredth in the same units of measurement (per NCES Standard 5-3). For example, a temperature of "about 55°C" should include any concentration that falls within 55°C ± 2.75°C.

[0056] C. Optional Washing Steps

[0057] The depigmentation method may optionally include one or more washing steps during the incubation period. 2 O 2 The bleach solution is removed from the sample and a wash buffer is deposited on the sample and incubated for a relatively short period of time. The wash buffer is then removed from the sample and H 2 O 2 A new aliquot of bleach solution is deposited onto the sample. If a washing step is included, the time taken by the washing step is considered part of the incubation period.

[0058] The wash buffer is typically a neutral buffered saline solution, which may also contain a small amount of detergent. Exemplary wash buffers include, for example, phosphate buffered saline (PBS), PBS-Tween 20, Tris buffered saline (TBS), TBS-Tween 20 (polysorbate 20), Tris-HCl, Tris-HC-Tween 20, phosphate buffer (PB), AP buffer, and the like.

[0059] III. AUTOMATED AFFINITY DYEING DEVICES AND METHODS

[0060] The present destaining method is particularly useful for automated affinity histochemical or cytochemical methods of staining cell samples (collectively referred to as "affinity staining"). Examples of affinity staining techniques include immunohistochemistry (IHC) and in situ hybridization (ISH). Affinity histochemical and cytochemical staining techniques typically involve contacting a sample, deposited on a slide or other solid support, with a biomarker-specific reagent under conditions sufficient to allow specific binding between the biomarker-specific reagent and the biomarker of interest. Binding of the biomarker-specific reagent to the biomarker facilitates deposition of a detectable moiety onto the sample near the location containing the biomarker. The detectable moiety can be used to identify and / or quantify the biomarker to which the biomarker-specific reagent is directed. The presence and / or concentration of the target in the sample can thereby be detected by detecting the signal generated by the detectable moiety.

[0061] Automated affinity stainers typically include at least reservoirs of the various reagents used in the staining protocol, a reagent dispensing unit in fluid communication with the reservoirs for dispensing the reagents onto the slides, a waste removal system for removing used reagents and other waste from the slides, and an environmental control system for regulating environmental factors (such as temperature, humidity, and / or pressure), and a control system for coordinating the action of the reagent dispensing unit, the waste removal system, and the environmental control system. In addition to performing the staining step, many automated affinity histochemical stainers can also perform (or are compatible with another system that performs) steps incidental to staining, such as slide baking (to adhere the sample to the slide), degreasing (also called deparaffinization), antigen retrieval, counterstaining, dehydration and clearing, coverslipping, etc. Overview of Automated Immunohistochemistry, Arch Pathol Lab Med., Vol. 138, pp. 1578-1582 (2014), which is incorporated herein in its entirety, describes several specific examples of automated IHC / ISH slide stainers and their various features, including INTELLIPATH (Biocare Medical), WAVE (Celerus Diagnostics), DAKO OMNIS and DAKO AUTOSTAINER LINK 48 (Agilent Technologies), BENCHMARK (Ventana Medical Systems, Inc.), LEICA BOND, and LAB VISION AUTOSTAINER (Thermo Scientific) automated slide stainers.Ventana Medical Systems, Inc. is the assignee of several U.S. patents disclosing systems and methods for performing automated analyses, including U.S. Pat. Nos. 5,650,327, 5,654,200, 6,296,809, 6,352,861, 6,827,901, and 6,943,029, as well as U.S. Patent Application Publication Nos. 20030211630 and 20040052685, each of which is incorporated herein by reference in its entirety. Commercially available staining units typically operate on one of the following principles: (1) open individual slide staining, where the slide is placed horizontally and reagents are dispensed as paddles onto the surface of the slide containing the tissue sample (as implemented, for example, in the DAKO AUTOSTAINER Link 48 (Agilent Technologies) and INTELLIPATH (Biocare Medical) stainers); (2) liquid overlay technique, where reagents are covered by or dispensed through an inert fluid layer deposited on the sample (as implemented, for example, in the BENCHMARK and DISCOVERY stainers (Roche)); (3) capillary gap staining, where the slide surface is placed close to another surface (such as another slide or a cover plate) to create a narrow gap through which capillary forces draw in and keep the liquid reagents in contact with the sample (as the staining principle used in the DAKO TECHMATE, LEICA BOND, and DAKO OMNIS stainers). Even if capillary gap staining is repeated several times, the fluids in the gap do not mix (e.g. in DAKO TECHMATE and LEICA BOND). A variation of capillary gap staining, called dynamic gap staining, uses capillary forces to apply the sample to the slide and then translates the parallel surfaces relative to each other during incubation to agitate the reagents and achieve reagent mixing (e.g. the staining principle implemented in the DAKO OMNIS slide stainer (Agilent)).In translational gap staining, a translatable head is placed above a slide. The underside of the head is spaced from the slide by a first gap small enough to allow a meniscus of liquid to form from the liquid on the slide during translation of the slide. A mixing extension having a lateral dimension smaller than the width of the slide extends from the underside of the translatable head to define a second gap smaller than the first gap between the mixing extension and the slide. During translation of the head, the lateral dimension of the mixing extension is sufficient to cause a lateral movement in the liquid on the slide generally in a direction from the second gap to the first gap. See WO 2011 / 139978. Recently, it has been proposed to use inkjet technology to deposit reagents on slides. See WO 2016 / 170008. This list of staining techniques is not intended to be comprehensive, and any fully or semi-automated system for performing biomarker staining may be incorporated into the histochemical staining platform.

[0062] When performed with an automated affinity stainer, H 2 O 2 The bleaching solution may be stored as a consumable on the device (such as in a reservoir in fluid communication with the dispenser). 2 O 2 Relatively low concentrations of H in the bleach solution 2 O 2 may not be stable enough for long-term storage in the device. Therefore, H 2 O 2 It may be preferable to generate the bleach solution in situ. Thus, for example, the device may be configured to generate a solution of concentrated H 2 O 2 The device may include a reservoir of preservation solution and a reservoir of buffer solution. The preservation solutions are then mixed in the device to form a H 2 O 2 A bleach solution is formed.

[0063] For example, a storage buffer (such as a Tris-based buffer, a citrate-based buffer, a phosphate-based buffer, etc.) may be deposited onto the sample, followed by deposition of concentrated H 2 O 2 The stock solution was diluted in the sample storage buffer and 2 O 2 A bleaching solution is obtained, after which the depigmentation process proceeds as described above.

[0064] In another example, the device may include a storage buffer, a storage concentrated H 2 O 2 The term "pH adjusting solution" includes a storage buffer, a storage concentrated H2O solution, and a storage pH adjusting solution. As used herein, a "pH adjusting solution" is a solution that can be used to adjust the pH of another solution. Examples include buffers of a defined pH (such as pH 9), such as Tris buffer, citrate buffer, or phosphate buffer. Storage buffer, storage concentrated H2O solution ... 2 O 2 solution, storage pH adjustment solution was deposited on the sample, and H 2 O 2 A bleaching solution is obtained, after which the depigmentation process proceeds as described above.

[0065] FIG. 1 shows an exemplary automated affinity staining workflow using a storage solution.

[0066] A. Degreasing

[0067] If the sample is in a wax block (e.g., a paraffin-embedded sample, e.g., a formalin-fixed, paraffin-embedded (FFPE) tissue sample), a delipidation step 101 is performed before the destaining method. Some automated affinity stainers can perform the delipidation step on the device. In such cases, the wax-embedded sample is placed directly on the device and a delipidation procedure (such as a deparaffinization process) is performed before the destaining method. Otherwise, the delipidation step is performed off-device and the delipidated sample is placed on the device. For other sample types (such as frozen sections), the delipidation step may be omitted.

[0068] B. Preservation solution and H 2 O2 Formation of the bleach solution

[0069] Storage solution (storage buffer, storage solution concentrated H 2 O 2 solution, and optionally a pH adjusting solution) together on a slide and 2 O 2 A bleaching solution 102 is obtained. In an embodiment, the stock solution concentrated H 2 O 2 The solution is a 4% to 20% H2SO4 solution in a diluent selected from the group consisting of water, a Tris-based aqueous buffer, a citrate-based aqueous buffer, or a phosphate-based aqueous buffer. 2 O 2 In another embodiment, the storage solution is concentrated H 2 O 2 The solution is 4% to 20% H in water. 2 O 2 In another embodiment, the storage solution is concentrated H 2 O 2 The solution is 5% to 20% H in water. 2 O 2 In another embodiment, the storage solution is concentrated H 2 O 2 The solution is 10% to 20% H in water. 2 O 2 In another embodiment, the storage solution is concentrated H 2 O 2 The solution is 5% to 15% H in water. 2 O 2 In another embodiment, the storage solution is concentrated H 2 O 2 The solution is 10% to 15% H in water. 2 O 2 In another embodiment, the storage solution concentrated H 2 O 2 The solution is 4% to 20% H 2 O 2 and 0 to 75 mM Tris. 2 O 2 The solution is 5% to 20% H 2 O 2 and 0 to 75 mM Tris. 2 O 2The solution is 10% to 20% H 2 O 2 and 0 to 75 mM Tris. 2 O 2 The solution is 5% to 15% H 2 O 2 and 0 to 75 mM Tris. 2 O 2 The solution is 10% to 15% H 2 O 2 and 0 to 75 mM Tris. 2 O 2 The solution is 4% to 20% H 2 O 2 and 10 to 75 mM Tris. 2 O 2 The solution is 5% to 20% H 2 O 2 and 10 to 75 mM Tris. 2 O 2 The solution is 10% to 20% H 2 O 2 and 10 to 75 mM Tris. 2 O 2 The solution is 5% to 15% H 2 O 2 and 10 to 75 mM Tris. 2 O 2 The solution is 10% to 15% H 2 O 2 and 10 to 75 mM Tris. 2 O 2 The solution is used with a storage buffer containing 10 to 100 mM Tris. 2 O 2 The solution is used with a storage buffer containing 10-100 mM Tris and a storage pH adjusted solution containing 50-500 mM Tris.

[0070] C. Incubation Period with Optional Washing Steps

[0071] The heating source may then be activated as necessary to obtain the temperature and period of time described in Section II (referred to as the incubation period 103). 2 O 2 Remove the bleach solution and wash the samples with wash buffer, then centrifuge for 102 h as described in 2 O 2 One or more optional wash and replenish cycles 104 may be performed during the incubation period 103 by regenerating the bleach solution. At the end of the incubation period 103, H 2 O 2 The bleaching solution is washed away with a wash buffer 105. The wash buffer is typically a neutral buffered saline solution that may also contain a small amount of detergent. Exemplary wash buffers include, for example, phosphate buffered saline (PBS), PBS-Tween 20, Tris buffered saline (TBS), TBS-Tween 20 (polysorbate 20), Tris-HCl, Tris-HC-Tween 20, phosphate buffer (PB), AP buffer, and the like.

[0072] D. Affinity staining

[0073] If the biomarker-specific reagent is an antibody or antibody-like specific detection reagent, it may be necessary to perform an epitope retrieval process 106 (also called antigen retrieval). Exemplary epitope retrieval processes include: heat-induced epitope retrieval (HIER), which involves heating the sample in various buffers at different pH levels; protease-based epitope retrieval (PBER), in which the sample is digested with proteolytic enzymes before staining; and a combination of HIER and PBER. Various specific epitope retrieval processes have been reviewed by Shi et al., D'Amico et al., Yamashita et al., Vinod et al., and Warford et al., but these are not exhaustive. Whether epitope retrieval is performed and the particular form of epitope retrieval used will depend on the particular biomarker-specific reagent selected and may need to be empirically determined for each biomarker-specific reagent used. A destaining method is typically performed prior to any epitope retrieval steps.

[0074] After epitope retrieval 106, detection reagents (including biomarker-specific reagents) are applied to affinity stain the sample 107. In a particular embodiment, the detection reagents are for immunohistochemical staining using DAB as a detectable moiety. In another example, the sample is a melanoma sample. The biomarker-specific reagents are antibodies against a biomarker selected from the group consisting of LAG3, AXL, TIM3, FAP, CD8, PD-1, PD-L1, SOx10, MART1 / MelanA, PRAME, HMB45, or CD8. The detection reagents deposit a chromogenic agent (such as DAB).

[0075] Non-limiting examples of commercially available detection reagents or kits containing detection reagents suitable for use in the methods of the invention include the following: VENTANA ULTRAVIEW Detection System (secondary antibodies conjugated to enzymes, including HRP and AP), VENTANA IVIEW Detection System (biotinylated anti-species secondary antibodies and streptavidin-conjugated enzymes), VENTANA OPTIVIEW Detection System (anti-species secondary antibodies conjugated to haptens and anti-hapten tertiary antibodies conjugated to enzyme multimers), VENTANA Amplification Kit (unconjugated secondary antibodies that can be used with any of the above VENTANA Detection Systems to amplify the number of deposited enzymes at the sites of primary antibody binding ... OPTIVIEW Detection System (anti-hapten secondary antibodies conjugated to haptens and anti-hapten tertiary antibodies conjugated to enzyme multimers), VENTANA OPTIVIEW Detection System (anti-hapten secondary antibodies conjugated to haptens and anti-hapten tertiary antibodies conjugated to enzyme multimers), VENTANA OPTIVIEW Detection System (anti-hapten secondary antibodies conjugated to haptens and anti-hapten tertiary antibodies conjugated to enzyme multimers), VENTANA OPTIVIEW Detection System (anti-hapten secondary antibodies conjugated to haptens and anti-hapten tertiary antibodies conjugated to enzyme multimers), VENTANA OPTIVIEW Detection System (anti-hapten secondary antibodies conjug amplification systems (include an anti-species secondary antibody conjugated to a hapten, an anti-hapten tertiary antibody conjugated to an enzyme multimer, and a tyramide conjugated to the same hapten. In use, the secondary antibody is contacted with the sample to allow binding to the primary antibody. The sample is then incubated with the anti-hapten antibody to allow association of the enzyme with the secondary antibody. The sample is then incubated with tyramide to allow attachment of additional hapten molecules. The sample is then incubated again with the anti-hapten antibody to allow attachment of additional enzyme molecules. The sample is then incubated with a detectable moiety to allow dye attachment; VENTANA DISCOVERY, DISCOVERY OMNIMAP, DISCOVERY ULTRAMAP anti-hapten antibody, secondary antibody, chromogen, fluorophore, and dye kits (each of which is available from Ventana Medical Systems, Inc., Tucson, AZ); POWERVISION and POWERVISION+ IHC Detection Systems (secondary antibodies directly polymerized with HRP or AP resulting in compact polymers with high enzyme to antibody ratios); and DAKO ENVISION+ System (enzyme-labeled polymer conjugated to secondary antibodies). In certain embodiments,

[0076] If desired, the affinity stain 107 may also include a counterstain to help identify morphologically relevant regions. Examples of counterstains include chromogenic nuclear counterstains such as hematoxylin (stains blue to purple), methylene blue (stains blue), toluidine blue (stains nuclei dark blue, polysaccharides pink to red), nuclear fast red (also called Kerneckrot stain, stains red) and methyl green (stains green); non-nucleogenic stains such as eosin (stains pink); 4',6-diamino-2-phenylindole (DAPI, stains blue), propidium iodide (stains red), Hoechst stain (stains blue), nuclear green DCS1 (stains green), nuclear yellow (Hoechst S769121, stains yellow at neutral pH and blue at acidic pH), DRAQ5 (stains red), DRAQ7 (stains red); and fluorescent non-nuclear stains such as fluorophore-labeled phalloidin (stains filamentous actin, color depends on the conjugated fluorophore). EXAMPLES

[0077] V. Working Examples Example 1: Lever of Hydrogen Peroxide Bleaching Process

[0078] Various depigmenting conditions were evaluated to identify the main factors in the bleaching process. Melanoma tissue was used as a model system. Formalin-fixed, paraffin-embedded multiple tissue blocks (MTBs) were prepared, each containing four melanoma cases and one non-neoplastic tonsil case.

[0079] Five variables were tested for their effect on depigmentation in melanoma tissue: (A) buffer composition [10 mM Tris and phosphate-buffered saline (PBS)]; (B) pH [pH 4, 7.4, or 9]; (C) pH [pH 4, 7.4, or 9]; (D) pH [pH 4, 7.4, or 9]; (E) pH [pH 4, 7.4, or 9]; (F) pH [pH 4, 7.4, or 9]; (G) pH [pH 4, 7.4, or 9]; (H) pH [pH 4, 7.4, or 9]; (I ... 2 O 2 concentration [1%, 10%, or 20% (w / w)]; (D) incubation temperature [37°C, 65°C, or 100°C]; and (E) incubation time [8 min, 48 min, or 92 min].

[0080] H 2 O 2 Stock solutions were obtained by diluting a 30% (w / w) solution in deionized water to either 1%, 10%, or 20%. Final concentrations were confirmed by titration with either potassium permanganate or cerium(IV) sulfate solutions. The diluted solutions were loaded into a reagent dispenser compatible with the BENCHMARK ULTRA automated slide stainer.

[0081] A BENCHMARK ULTRA automated slide stainer was programmed to run the protocol described in Table 1. [Table 1]

[0082] The “bleaching” protocol included the following steps: (1) dispense a buffer paddle [reaction buffer (Roche)] onto the slide; (2) add 0.25%, 2.5%, or 5% final H 2 O 2 Concentration: Buffer:H 2 O 2 Add H to the paddle in a v / v ratio of approximately 3:1. 2 O 2 (3) dispense liquid coverslip over the paddle; (4) incubate the sample at the listed temperature for the listed period of time; and (5) wash the slide after the listed period of time.

[0083] Bleached slides were scored for overall melanin intensity, melanin coverage and nonspecific background. Based on the score provided, each condition was given a rank of 1 to 5, with 1 indicating the best bleaching performance and preservation of tissue integrity and 5 indicating the worst bleaching performance and / or preservation of tissue integrity. A description of each score is provided in Table 2. [Table 2]

[0084] All test conditions and their associated scores are shown in Table 3: [Table 3]

[0085] Significant tissue loss was observed at an incubation temperature of 100°C and an incubation time of 92 minutes. 2 O 2 The bleaching performance was insufficient with the storage solution.

[0086] Conditions 3, 4, 10, and 17 resulted in sufficient bleaching without damaging tissue morphology. Therefore, samples bleached under these conditions were immunohistochemically stained with LAG3 monoclonal antibody (clone SP464; Ventana Medical Systems, Inc.). Representative images of IHC-stained tissues are shown in Figure 2. Condition 3 (Tris pH 9, 20% HO) 2 O 2 , 37 °C, 92 min) yielded the best results compared to other conditions tested: sufficient bleaching, epitope preservation with LAG3 stained slides was observed, and cell morphology was not damaged by the bleaching process.

[0087] The rankings in Table 3 were analyzed in MINITAB 18 (Minitab, LLC) for the effect of bleaching conditions on overall performance. The results are shown in Figures 3A-3C. The combination of incubation time and temperature had the greatest effect on overall performance (Figure 3A). The bar for the combination of time and temperature (DE) crosses the baseline at 2.23, indicating that this coefficient is statistically significant at the 0.05 level for the current model terms. Increased time, especially in combination with high temperature, contributed to poor morphology. Buffer and pH were significantly associated with H 2 O 2Compared to concentration, time and temperature, it had the least effect on overall performance. The main effects plot in Figure 3B shows that buffer composition had the least effect on overall performance, while incubation temperature had the most effect. Bleaching performance appeared to be better at pH 9 compared to pH 4 and 7.4. The 20% H 2 O 2 The 1% and 10% concentrations resulted in the best bleaching compared to 1% and 10%. As temperature and time increase, the bleaching performance worsens. As can be seen in the interaction plot in Figure 3C, higher H 2 O 2 The concentrations (10% and 20%) were ranked best at the lower temperature (37° C.), while the lower concentration (1%) was ranked best at 65° C. The lower temperature (37° C.) was ranked best at the longer time (92 min), but performance was poorer at the medium and high temperatures (65° C. and 100° C.).

[0088] Example 2: Optimization of concentration, time, and temperature of 3x3x3

[0089] A 3 × 3 × 3 study was conducted with three factors and levels listed in Table 4. [Table 4]

[0090] The bleaching reagent is H 2 O 2 Stock solutions were prepared by diluting to the desired test concentrations with 10 mM Tris, pH 9. Using the staining protocol in Table 5 and Tris, pH 9 as the buffer paddle, a single MTB slide was bleached in each of the 27 conditions in Table 5. [Table 5]

[0091] Additional slides bleached in conditions 6, 12-14, and 20-22 were stained for LAG3. 8.1.2.4 The data was analyzed and plots in MINITAB 18 were generated using the ranking system described in Table 2.

[0092] The Pareto plot in Figure 4A demonstrates that the most influential factor on overall performance is incubation temperature. The bar for temperature (B) crosses the reference line at 2.08, indicating that this coefficient is statistically significant at the 0.05 level for the current model terms. As temperature increased, there was a corresponding increase in slide acceptability due to morphological damage.

[0093] As can be seen in the main effects plots in Figure 4B, bleaching performance was significantly improved for all three concentrations of H 2 O 2 The bleaching performance was similar for all three incubation times, with 10% ranking slightly better than 1% and 20%. Bleaching performance for all three incubation times was comparable. Incubation temperature had the greatest effect on performance, with higher temperatures ranking worse.

[0094] The interaction plot in Figure 4C shows that lower temperatures consistently ranked better across concentrations, whereas higher temperatures ranked worse across concentrations. The interaction of concentration with time was more gradual than concentration compared to temperature. Higher temperatures performed poorly regardless of time.

[0095] The results suggested that lower temperatures were better for uniformity. Furthermore, 10% and 20% H 2 O 2 The bleaching performance of the two solutions was not significantly different. However, the lower concentration (1%) did not produce sufficient bleaching.

[0096] Example 3: Optimization of concentration and time at 37°C and 50°C

[0097] The purpose of this study was to determine the H 2 O 2 The concentration and incubation time were further optimized. 2 O 2 Stock solutions were prepared by diluting to the desired test concentrations with 10 mM Tris, pH 9. Using the staining protocol in Table 1 and 10 mM Tris, pH 9 as the buffer paddle, a single MTB slide was bleached in each of the 48 conditions in Table 6. [Table 6]

[0098] 10% H for 64 min at 50 °C 2 O 2 The bleaching performance produced by 6% H2O at 50 °C for 80 min was 2 O 2 and 5% H for 92 min at 50°C. 2 O 2 Representative images from this study are shown in Figure 5.

[0099] 10% H for 64 min at 50 °C 2 O 2 50°C conditions resulted in sufficient bleaching while preserving morphology. However, compared with 37°C, the 50°C condition resulted in nuclear damage in some tissues. This effect on tissue morphology at 50°C was not significantly different from that at 5% H 2 O 2 and 10% H 2 O 2 was observed at 92 minutes.

[0100] 10% of H 2 O 2 Samples bleached with 10% H for 92 min at 37°C showed virtually complete reduction of melanin pigment in the case of highly pigmented MTB tissue, while preserving tissue morphology. Amelanotic melanoma cases showed concordant LAG3 immunocytostaining with or without bleaching, confirming epitope preservation. Based on these results, 10% H incubated for 92 min at 37°C was used to determine the epitope preservation. 2 O2 was selected as the nominal bleaching condition for subsequent testing.

[0101] Example 4: Additional Time and Temperature Evaluations

[0102] Our previous findings from Example 3 showed that under nominal conditions (10% H 2 O 2 Our results show that bleaching at 200 °C for 92 min (37 °C) promotes the removal of melanin while preserving tissue LAG3 expression as well as cell morphology. To identify other potential conditions with efficient bleaching capabilities, various conditions described in the literature were tested (Foss, McGovern, Shen, and Orchard (2007)). Bleaching performance (i.e., percentage of remaining melanin, melanin staining intensity), melanoma histomorphology, and tonsillar histomorphology were evaluated and further compared to reference (nominal) conditions.

[0103] Four melanoma MTBs were used in further optimization of incubation times and temperatures. Slides were bleached under the conditions listed in Table 7. [Table 7]

[0104] The bleaching reagent is H 2 O 2 Stock solutions were prepared by diluting with deionized water to the desired test concentrations. One slide per MTB was bleached for each condition followed by IHC staining with LAG3 clone SP464 or NRC. Acceptability was assessed retrospectively using the criteria outlined in Table 2. Results are shown in Table 8. [Table 8]

[0105] All slides showed sufficient bleaching to allow interpretation of the specific LAG3 DAB signal. Furthermore, tissue antigenicity was preserved in all slides. Overall bleaching performance and LAG3 signal were comparable to nominal conditions (10% H in DI water). 2 O 2 , 37℃, 92 minutes).

[0106] Higher incubation temperatures (50°C and 65°C) resulted in better bleaching performance, but higher temperatures also showed a higher incidence of mild to moderate morphological damage.

[0107] 20% of H 2 O 2 Slides bleached for 60 min at 37 °C with H produced acceptable morphology relative to nominal conditions, with slightly improved bleaching performance. 2 O 2 Increasing the concentration of H to 30% had a detrimental effect on cell morphology. 2 O 2 The smallest improvement in bleaching performance observed was observed in the nominal formulation H 2 O 2 did not justify an increase in the concentration of

[0108] Example 5: Evaluation of long incubation times

[0109] The aim of this study was to evaluate the upper limit of bleaching incubation time and its effect on tissue morphology and counterstain acceptability. To evaluate cell morphology and counterstain performance, three MTBs were stained in duplicate with NRC after the bleaching step. Two slides per MTB were stained with 10% H2O diluted in DI water. 2 O 2 The slides were bleached at 4 °C for 1 h and incubated at 37 °C for the times indicated in Table 9. For each bleaching condition, slides were considered unacceptable if there was loss of tissue integrity and / or nuclear damage, as demonstrated by weak counterstaining. In all cases, replicate slides were comparable to each other. [Table 9]

[0110] At 120 min, all tissues in all MTBs were acceptable, comparable to the nominal 92 min condition. A small number of cases of morphological and / or counterstain intolerance were observed in K and R MTBs at the 180 min incubation time. By 240 min, all MTBs showed tissue intolerance in at least one tissue. At 300 min, 9 of 10 tissues across the three MTBs showed tissue intolerance. See Table 10 for a summary of the results of this study. Representative images are shown in Figure 6. [Table 10]

[0111] Example 6: Evaluation of diluent composition and paddle conditions

[0112] The purpose of this study is to determine the amount of Tris and PBS buffers that can be used in H 2 O 2 The objective of this study was to determine whether the pH / buffering of the diluent would affect bleaching performance compared to deionized water as the diluent. A buffer solution was used as the diluent to achieve alkalinity for the bleaching reaction. However, the H 2 O 2 The concentration of H is high (10% w / w, approximately 3.2M) and can determine the pH of the formulated product. Therefore, to understand the pH of the formulated product, 32% H 2 O 2 pH measurements were performed by diluting to 10% with the desired diluent and then measuring the pH with a pH meter. The results are shown in Table 11. [Table 11]

[0113] 1x PBS and 10 mM TrisHO 2 O 2 The pH analysis of the high concentration of H2 O 2 It has been found that the pH of the final bleaching reagent solution is determined by the pH of the solution. Therefore, the buffers used as diluents do not operate within their respective buffering capacities, but rather by the pH of the solution. 2 O 2 The pH was reduced from the alkaline range. Bleaching performance was similar across all formulations tested (see Table 12 for a summary of results and Figure 7 for representative images). In all evaluable bleached slides, the residual melanin had an overall intensity that allowed interpretation of LAG3-specific staining. Furthermore, LAG3 staining was consistent across all evaluable bleached slides. In summary, H 2 O 2 Water was used as the diluent with similar results being obtained with other diluents. [Table 12]

[0114] The chemistry of the reaction paddle on the slide while it was on the slide was then examined using various bleaching reagent formulations and pH-adjusted buffers (diH2SO4 at pH 9). 2 The bleaching reagent diluents (1x PBS, 10 mM Tris, DI water, CC1 and reaction buffer), 10% HO in those diluents, 2 O 2 formulations, and paddle formulations (10% H in their diluents in reaction buffer paddle 2 O 2 ;2.5% H 2 O 2 pH measurements of the final "on-slide" concentration of 100 mg / ml were performed at various temperatures.

[0115] Non-automated bleaching procedures (such as those of Orchard (2007) and Chung) suggested that alkalinity of the reaction solution could improve bleaching performance. Therefore, the paddle composition on the slide was replicated in an offline environment and the pH was assessed after successive applications of pH-adjusting buffers. DI water was used as the diluent to assess the pH. 2 O 2Various formulations of were prepared: 10% H without pH adjustment. 2 O 2 Based on this, 12.5% ​​of H 2 O 2 and 15% H 2 O 2 were formulated for use in sequential applications, either as a single or dual application of pH-adjusted buffer. The concentrations of pH-adjusted buffer used in single applications ranged from 50 mM to 500 mM Tris, and for dual applications, 450 mM or 500 mM Tris was used. The final "on-slide" concentration of all formulations was 2.5% H 2 O 2 The results are shown in Table 13 and Figure 8. [Table 13-1] [Table 13-2]

[0116] These results indicate that the addition of pH-adjusted buffer to the paddles can create alkaline conditions for the bleaching step. Therefore, we evaluated additional dispensing of pH-adjusted buffer (250 mM or 500 mM Tris) to the paddles before dispensing the bleaching reagent. Furthermore, the effect of paddle composition on bleaching performance was evaluated by replacing the nominal reaction buffer (Tris-based buffer with BRIJ-35 surfactant, pH 7.6) paddles with other bulk reagents, such as CC1 (Tris-borate-EDTA-based buffer, pH 8.55), EZ Prep (aqueous composition of PROCLIN300 biocide and COLATERGE low foam surfactant), or SSC (sodium chloride citrate-based buffer, pH 7).

[0117] Three MTB single slides were incubated with reaction buffer, EZ Prep and CC1 paddles and pH adjustment buffer (250 mM or 500 mM Tris) was applied, followed by 12.5% ​​H2O in Tris pH 9. 2 O 2 (Final concentration on slide: 2.5% H2 O 2 )) for 92 minutes at 37°C. A single slide of MTB E was also tested by application of reaction buffer, CC1 and EZ Prep paddles and pH adjustment buffer (250 mM Tris only), followed by 12.5% ​​H2O in water. 2 O 2 The test was performed for 92 minutes at 37°C with application of 100 mM Tris pH buffer. The test was performed in duplicate on three additional MTB. In addition to the reaction buffers, CC1 and EZ Prep, SSC was also evaluated as a reaction paddle during this test. After testing 500 mM Tris pH adjustment buffer, 12.5% ​​H2O in water was used. 2 O 2 was applied for 92 min at 37°C. For these MTB, bleached NRC was also included in each condition tested. All conditions evaluated were compared to the nominal bleaching condition: 10% H 2 O 2 Without applying the pH adjustment buffer, add 300 µL of paddle reaction buffer (final on-slide concentration 2.5% H 2 O 2 The results are shown in Table 14. [Table 14-1] [Table 14-2]

[0118] Regardless of the MTB, reaction paddles with SSC and pH-adjusted buffer resulted in washout of either NRC or LAG3 staining (or both) in all melanoma tissues in all three MTBs. Furthermore, the use of EZ Prep or CC1 as reaction paddles resulted in the loss of at least one tissue in each of the MTBs. The nominal bleaching conditions only showed tissue loss under circumstances where there was universal loss associated with that tissue across all conditions tested. Furthermore, reaction buffer paddles dispensed with pH-adjusted buffer showed tissue loss in the other MTB in addition to tissue shredding in one MTB. These results suggest that reaction buffer is more suitable as a reaction paddle for the bleaching process compared to CC1, SSC or EZ Prep.

[0119] For slides bleached under nominal conditions, LAG3 IC staining was consistent (≦0.5 points) compared to no bleach slides, where applicable.

[0120] The LAG3 IC staining intensity of hypermelanotic cases in MTB6 bleached with pH-adjusted buffer was 1 point lower compared to nominal conditions, and there was a 1 point decrease in tonsillar IC staining intensity in MTB F2 bleached with pH-adjusted buffer compared to slides without bleaching.

[0121] Comparing the high melanin cases, bleaching with pH-adjusted buffer resulted in a greater decrease in melanin coverage (approximately 20%) in the two MTBs. However, melanin depigmentation performed in the reaction buffer paddle with or without the addition of pH-adjusted buffer was sufficient for interpretation of LAG3. These results suggest that the nominal depigmentation conditions result in sufficient depigmentation for interpretation of LAG3. Furthermore, modifying the configuration of the system to add additional pH-adjusted buffer may improve residual melanin but may also have a detrimental effect on the tissue and LAG3 staining.

[0122] Finally, compared to the application of pH-adjusted buffers (250 mM or 500 mM), 10% H2SO4 formulated in either deionized water or 10 mM Tris2 O 2 The impact of diluent composition was evaluated by a direct comparison of Four replicate slides of melanoma MTB were tested under the conditions listed in Table 15. [Table 15]

[0123] A pH adjustment buffer (250mM or 500mM Tris) was applied to the reaction buffer paddle (where applicable), followed by bleaching reagent and incubation for 92 minutes at 37°C. All conditions were compared to nominal bleaching conditions. 10% H2O diluted in water was added. 2 O 2 Dispense in a single aliquot of 300 μL of reaction buffer, with an effective concentration of 2.5% H 2 O 2 To add the pH adjustment buffer, 2 O 2 was formulated at 12.5% ​​to have the same active concentration of 2.5% in the paddle compared to the nominal condition. The pH of each formulation at room temperature was measured offline. The bleaching procedure was modified to accommodate the dispensing of additional pH adjustment buffer in the bleaching reaction paddle. The results are shown in Table 16. [Table 16]

[0124] All bleaching conditions tested showed similar bleaching performance and LAG3 expression in moderate and low / no melanin cases across all enrolled MTBs. 2 10% HO + 500 mM Tris 2 O 2The case of high melanin expression in MTB2-4 bleached with " demonstrated complete removal of melanin in at least one of the duplicate LAG3 stained slides, compared to the incomplete but acceptable depigmentation observed in the other conditions tested. However, this slight improvement in bleaching performance did not improve the ability to read the specific LAG3 staining, as LAG3 expression was interpretable across all tested conditions. IC staining in one of the tissues from MTB2 was 1 pt, and was not significantly different from the 10% H in DI water bleached cases. 2 O 2 In 100% of the 15 melanomas, 10% were IC staining, whereas in 20% were IC staining. There is no control for IC in this case, since non-bleached slides are not evaluable for IC staining. However, 14 of 15 melanomas across the 4 MTBs showed identical IC staining across all 4 formulations. Furthermore, readers scored IC staining in 1-point increments. Thus, a 1-point difference between formulations was considered equivalent.

[0125] Example 7: Bleaching effect before and after antigen retrieval

[0126] Additional studies were performed to evaluate the impact of antigen retrieval before and after bleaching. Two MTBs were tested. One slide per MTB was bleached (CC1 for 64 minutes) either before or after antigen retrieval and subsequently stained with LAG3 or rabbit monoclonal negative control Ig. One slide per MTB was also stained with LAG3 without bleaching to serve as a non-bleached control. The results are shown in Tables 17 and 18. [Table 17] [Table 18]

[0127] Bleaching performance was considered acceptable across all tissues bleached pre-CC1, except for one tissue (tissue 1, MTB2), which was considered unacceptable because the bleaching reagent reduced melanin by only 0.75 points across all melanin intensities and only 5% of the area percentage when compared to the unbleached reference. Bleaching performance in tissues bleached after antigen retrieval was considered unacceptable in tissues with moderate to high melanin levels; residual melanin was unchanged, less than 1.0 point; or 15% different compared to the unbleached reference slide, and / or LAG3 staining was not assessable through residual melanin. Minimal changes were observed across both MTBs in terms of both immune cell staining intensity and percent of immune cells stained between pre-bleach and post-bleach CC1 conditions. Two bleached pre-CC1 cases (tissues 2 and 3 of MTB2) showed a 0.25 point increase in immune cell staining intensity over their bleached post-CC1 counterparts. Tissue 4 of MTB2 was the only case that showed a difference in percent immune cell staining, which increased by 1% over bleaching after CC1. Background staining was acceptable across all tissues.

[0128] Example 8: Continuous bleaching

[0129] The objective of this study was to evaluate bleaching performance when a washing step was incorporated into the bleaching step and whether the washing step improved bleaching performance at high temperatures.

[0130] For evaluation of washing between successive bleaching steps, slides were incubated with the respective formulation of bleaching reagent for 44 min. Slides were then rinsed with reaction buffer and adjusted to optimal slide volume with reaction buffer. The respective bleaching reagent was then reapplied and slides were incubated for another 44 min. The 44 min incubation time was chosen to make the total incubation time comparable to the nominal time of 92 min. One slide was each bleached with and without LAG3 clone SP464 after the bleaching process. Table 19 summarizes all reaction conditions tested and the corresponding results. [Table 19]

[0131] H 2 O 2 Severe morphological damage was observed at 80°C, regardless of concentration or washing step. In addition, some morphological damage and loss of antigenicity was seen in some tissues at 60°C. There was no significant difference in bleaching performance between slides incubated for 92 min and those bleached with an intermediate washing step. 2 O 2 Although the study sufficiently removed melanin to easily assess LAG3 signal, melanin removal was not observed in the 5% and 1% H 2 O 2 Compared to the concentration, 10% H 2 O 2 The bleaching was nearly complete in slides bleached with 5% H in Tris pH 9. 2 O 2 Slides bleached for 92 min at 50 °C with 10% H produced bleaching and LAG3 staining comparable to nominal conditions (10% H 2 O 2 , 37℃, 92 minutes).

[0132] Example 9: Stability

[0133] The aim of this study is to investigate the effect of 10% H2O2 in various formulations using a thermal stress model. 2 O 2The aim of the study was to access stability.

[0134] Using the heat stress model, H 2 O 2 Accelerated stability studies were performed on multiple formulations of. This was accomplished by storing formulated reagent dispensers (used for functional testing) and formulated reagents in 100 mL bottles (for analytical testing) at elevated temperatures for extended periods of time. See Table 20 for formulation, temperature, time points, and sample details. [Table 20]

[0135] Accelerated stability testing was first performed using H 2 O 2 The five formulations were tested at 30°C, 37°C and 45°C for 45 days. 2 O 2 To further access the degradation of, two formulations stored at 60 °C for 30 days were also tested. Dispensers and reagent bottles were kept in incubators at 30 °C, 37 °C, 45 °C and 60 °C until the day of testing. After removal for testing, dispensers and bottles were stored at 2-8 °C until the completion of the study. Dispensers and bottles formulated on day 0 were stored at 2-8 °C for the duration of the study and were used as the reference for each time point. At each time point, functional tests were performed to evaluate bleaching and LAG3 assay performance. For each condition and time point, a nominal bleaching protocol of 92 min at 37 °C was used. Analytical tests (pH measurements and titrations) were also performed to evaluate the changes in pH values ​​and H2O2 concentrations relative to day 0.

[0136] Cerium (IV) sulfate titration was used to measure the concentrations of both stressed and non-stressed formulated products at each time point, and pH measurements of test reagents were performed using a calibrated pH meter.

[0137] At each time point, slides were stained with LAG3 (SP464) after bleaching with each heat-stressed bleaching reagent. A single slide was bleached with the non-stressed bleaching reagent of each formulation (stored at 2-8 °C) to serve as a reference. Melanin intensity, melanin coverage as well as LAG3 staining intensity and IC percentage were captured.

[0138] The pH value of the bleaching solution is directly dependent on the temperature at which it is measured. In Test 1, the heat-stressed reagents were not brought to room temperature before pH measurements were taken. Therefore, the ΔpH methodology (normalizing pH values ​​to the theoretical day 0 pH) was used to compare the change in pH across all time points. The results are shown in Figure 9 and Table 21. [Table 21]

[0139] The concentration of hydrogen peroxide in each solution was determined using cerium(IV) sulfate redox titration and is listed in Table 22. The change in concentration over time is shown in Figure 10. 2 O 2 It includes a reference line (red, dashed line) at [Table 22]

[0140] HO in both Tris and water formulations at 2-8°C. 2 O 2 The concentrations were stable for 45 days. The formulations showed no signs of decomposition by 32 days when stored at 30°C and 37°C. 2 O 2 The concentration remained constant (approximately 10%) for 45 days at 37° C. and 45° C. On day 45, the Tris solution was adjusted to %H at 37° C. and 45° C., respectively. 2 O 2 The decrease in H in Tris was about 0.5% and about 1.1%. 2 O 2The degradation of H became more pronounced (at 45°C) by day 60. The reaction buffer and PBS solutions showed a decrease in H by day 10 when stored at 30°C. 2 O 2 This shows a 0.7% decrease in concentration of H in PBS. 2 O 2 This gradual decline in H became more pronounced when stored at 37° C. and 45° C. The reaction buffer and PBS solutions showed a decrease in H by 10 days when stored at 30° C. 2 O 2 The concentration of H in PBS decreased by 0.7%. 2 O 2 This gradual decline in activity became more pronounced when stored at 37°C and 45°C.

[0141] The results of the functional tests are summarized in Table 23. [Table 23-1] [Table 23-2] [Table 23-3]

[0142] On day 3, 10% HO in PBS pH 8 stored at 45 °C 2 O 2 showed a slight decrease in melanin bleaching compared to the water and Tris pH 9 diluents. However, this slight increase in melanin signal was not reproduced at the 5, 8, or 10 day time points. Furthermore, H diluted in CC1 at day 0 with MTB P, S, and T showed a significant increase in melanin bleaching compared to the other bleaching reagent formulations. 2 O 2 Furthermore, on day 5, there was an increase in melanin intensity associated with H in CC1 stored at 30°C. 2 O 2 is another H 2 O 2 It showed a 1 point increase in melanin intensity over the formulation, but this observation was not repeated on day 10.

[0143] The Arrhenius model presumably requires that the assay fails when LAG3+ cells are no longer observed or the epitope is damaged. No failure was observed in any formulation by day 10, so testing was continued through day 45. H formulated in Tris pH 9 and DI water 2 O 2 showed that H is the most stable 2 O 2 Based on the titration data, these two formulations were further tested at days 32 and 45. Both formulations (Tris pH 9 and DI water) demonstrated concordant (IC SI of ≦0.5 points) melanin bleaching and LAG3 staining, with near complete bleaching in the hypermelanin cases. By day 45, no evidence of dysfunction (LAG3 epitope binding) or H 2 10% HO or Tris-9 diluted 2 O 2 No differences in melanin intensity were observed that would have prevented LAG3 interpretation using the 100% ... 2 O 2 are H at 37°C and 45°C, respectively. 2 O 2 The concentration of H in Tris pH 9 decreased by approximately 0.5% and approximately 1.1%. 2 O 2 The decomposition of becomes more pronounced by day 60 (at 45°C) and the water is converted to H 2 O 2 It was shown to be the most stable diluent.

[0144] Example 10: Guardbanding

[0145] The objective of this study was to evaluate the acceptability of formulation and protocol selectable: 2 O 2 The concentration, bleaching incubation temperature, and incubation time were to be determined.

[0146] Three MTBs were enrolled and replicate slides from each MTB were cultured at various H 2 O2 Concentrations, incubation temperatures, and incubation times were subjected to subsequent staining with LAG3 clone 17B4. One slide was bleached for each test condition and then stained with NRC. See Table 24 for details of test conditions. [Table 24]

[0147] Example 10AH 2 O 2 Concentration of

[0148] H 2 O 2 The guardbanding results are shown in Table 25 and Figures 11A-11D. [Table 25]

[0149] All slides showed sufficient bleaching to allow interpretation of the specific LAG3 DAB signal. Furthermore, expression was preserved in all slides compared to the non-bleached reference. After bleaching in the test conditions, all evaluable tissues showed concordant LAG3 staining compared to the non-bleached reference slide. Figures 11A and 11B show the distribution of melanin intensity and coverage in the five hypermelanotic cases. 2 O 2 Increasing the concentration to 17% and 20% resulted in unacceptable tissue damage / poor cell morphology in the hypermelanotic case of MTB3. Furthermore, localized morphological damage was also noted by the reader in one of the tissues at 17% and 20% MTB W1, but was nevertheless considered acceptable by the reader.

[0150] 1% to 15% H 2 O 2 The concentrations yielded sufficient melanin bleaching and LAG3 staining performance was consistent with no bleaching slides in all tissues from all three MTBs. 15% yielded almost complete removal of melanin, while 8%–12% H2 O 2 show similar levels of residual melanin, see Figures 11C and 11D.

[0151] Example 10B: Incubation Temperature

[0152] All slides bleached with the parameters listed in Table 24 demonstrated sufficient bleaching to allow interpretation of the specific LAG3 DAB signal. Where applicable, all tissues showed agreement (within 0.5 points) with the no-bleach slides. See Table 26 for a summary of results and Figure 12A for representative images from the study. [Table 26]

[0153] Figures 12B and 12C show the distribution of melanin intensity and coverage in five hypermelaninous cases of MTB1-3. Bleaching performance at room temperature, 37°C, and 45°C was comparable, with IC staining within 0.5 points versus no bleach slides. However, slides incubated at 37°C and 45°C showed a 0.5-1.5 point reduction in melanin intensity for residual melanin. Melanin coverage was reduced by 10-15% when compared to slides incubated at room temperature. LAG3 staining in tissues bleached at 55°C was consistent (within 0.5 points) with no bleach slides for MTB 1 and 3, whereas two of the five melanoma tissues associated with MTB2 showed either damaged tissue morphology or poor counterstaining.

[0154] At 65°C and 75°C, melanoma and tonsil samples from all three MTBs showed extensive cell damage and tissue washout. Across all MTBs, 13 of 30 tissues showed unacceptable tissue morphology, while 12 of 30 tissues were washed out during the bleach / stain run. Results indicate that incubation temperatures from room temperature up to 45°C result in sufficient bleaching without compromising staining performance and tissue integrity. However, melanin removal is more pronounced at incubation temperatures of 37°C and 45°C.

[0155] Example 10C: Incubation Time

[0156] All slides bleached with the parameters listed in Table 24 demonstrated sufficient bleaching to allow interpretation of the specific LAG3 signal. Additionally, tissue antigenicity was preserved as LAG3 staining for all tissues was within 0.5 points from the no-bleach slides, except for tissue 3 of MTB3, which showed a 0.75 point increase in LAG3 staining intensity at all incubation times compared to the non-bleached slides.

[0157] Figures 13A and 13B show the distribution of melanin intensity and coverage in five hypermelanotic cases of MTB1 to 3. See Table 27 for a summary of the results and Figure 13C for representative images of the study. [Table 27]

[0158] These data were obtained by bleaching the tissue with a nominal bleaching agent formulation (10% H 2 O 2) for 64 and up to 180 minutes provided sufficient bleaching while maintaining LAG3 staining performance. However, the qualified reader commented that at 180 minutes, there was focal morphological damage to two melanoma tissues and the tonsil in MTB1. Nevertheless, this reader considered the slides acceptable.

[0159] References Chung et al., A melanin-bleaching methodology for molecular and histopathological analysis of formalin-fixed paraffin-embedded tissue, Laboratory Investigation, 2016, Vol. 96, pp. 1116-1127.

[0160] Foss, A. et al., Immunohistochemical techniques: the effect of melanin bleaching, Br.J.Biomed.Sci., 1995, Vol. 52, Issue 1, pp. 22-25.

[0161] Hu, L. et al., Int. J. Clin. Exp. Pathol. (2020) 13(8), 2027-2034

[0162] Liu et al.,Melanin Bleaching With Warm Hydrogen Peroxide and Integrated Immunohistochemical Analysis:An Automated Platform,International Journal of Surgical Pathology,2018,Vol.26,Issue 5,410-416.

[0163] McGovern&Crocker,The Effect of Melanin Pigment Removal on the Peroxidase-Antiperoxidase Immunoperoxidase Technic,Am.J.Clin.Pathol.(1987),Vol.88,Issue 4,pp.480-483.

[0164] Manicam,C.et al.,Effective Melanin Depigmentation of Human and Murine Ocular Tissues:An Improved Method for Paraffin and Frozen Sections,PLOS One(2014)9(7),e102512.

[0165] Orchard,Use of heat provides a fast and efficient way to undertake melanin bleaching with dilute hydrogen peroxide,Br.J.Biomed.Sci.2007,64(2),89-91

[0166] Orchard et al.,Semi-automated standardisation of melanin bleaching procedures of heavily pigmented melanocytic lesions for immunohistochemical analysis on an automated platform,Br.J.Biomed.Sci.(2019),76(4),172-177.

Claims

1. A method for bleaching melanin from a cell sample, comprising the steps of: ２ O ２ H containing ２ O ２ A method comprising contacting the cell sample with a bleaching solution and incubating at a temperature between 20°C and 50°C for a period ranging from 8 minutes to 180 minutes.

2. 2. The method of claim 1, wherein the temperature is between 30°C and 50°C.

3. The H ２ O ２ 10. The method of claim 1, wherein the bleaching solution has a pH in the range of pH 7 to pH 9 at room temperature.

4. The H ２ O ２ 10. The method of claim 1, wherein the bleaching solution comprises up to 250 mM Tris.

5. 10. The method of claim 1, wherein the period of time ranges from 60 to 180 minutes.

6. ● The above H ２ O ２ The bleaching solution contains 1% to 5% hydrogen peroxide and 0 mM to 75 mM Tris; the temperature is in the range of 37°C to 50°C; said period of time is in the range of 60 to 180 minutes; The method of claim 1.

7. 1. An automated method for affinity staining a melanocyte sample, comprising: an automated affinity staining device having at least the following functions: (a) subjecting the melanocyte sample to the method of any one of claims 1 to 6 to obtain a bleached sample; (b) performing an antigen retrieval procedure on the bleached sample to obtain a conditioned sample; and (c) affinity staining the prepared sample with a set of detection reagents to obtain a stained sample; The method includes:

8. The automated affinity staining device includes a reservoir containing a storage buffer and a reservoir containing a concentrated hydrogen peroxide stock solution at a concentration ranging from 4% to 20%; ２ O ２ 8. The method of claim 7, wherein the aqueous solution is obtained by mixing a volume of the hydrogen peroxide stock solution with a volume of the buffer solution.

9. 8. The method of claim 7, wherein (b) includes a washing step during incubation, and the aqueous solution of hydrogen peroxide is replenished after said washing step.

10. 8. The method of claim 7, wherein the set of detection reagents comprises biomarker-specific reagents specific for a polypeptide selected from the group consisting of LAG3, AXL, TIM3, FAP, CD8, PD-1, PD-L1, SOx10, MART1 / MelanA, PRAME, HMB45, and CD8.

11. A stock melanin bleaching reagent for use in an automated affinity stainer, comprising a pH of 7.4-9.0 and a concentration of 4%-20% H ２ O ２ A stock melanin bleaching reagent, including an aqueous solution.

12. An automated affinity staining machine, comprising: (a) Concentrated H in concentrations ranging from 4% to 20% ２ O ２ a reagent set including a storage solution, a buffer, optionally a pH adjusting buffer, and optionally a washing buffer; and (b) a control system programmed to instruct the automated affinity staining machine to perform a set of operations on melanin pigmented tissue, the set of operations comprising: (b1) at least the concentrated H ２ O ２ The combination of the storage solution, the buffer, and optionally the pH-adjusted buffer provides a 1% to 5% H ２ O ２ H containing ２ O ２ Obtaining a bleaching solution; and (b2) Said H ２ O ２ Incubating the cell sample contacted with the bleaching solution for a period ranging from 8 minutes to 180 minutes at a temperature of 20°C to 50°C, the incubation optionally including at least one wash with the wash buffer, and ２ O ２ a control system including incubating, wherein the bleaching solution is replenished after said washing; Automated affinity staining machines, including:

Images