Biological sample staining system and method

Abstract

To provide a system and method for treatment of biological samples.SOLUTION: An automated biological sample staining system is provided, comprising at least one microfluidic reagent applicator, at least one bulk fluid applicator, at least one fluid aspirator, at least one sample substrate holder, at least one relative motion system, and a control system programmed to execute at least one staining protocol on a sample mounted on a substrate held by the at least one sample substrate holder.SELECTED DRAWING: Figure 10

Description

Technical Field

[0001] Cross-reference to Related Applications

[0001] This disclosure claims the benefit of U.S. Provisional Patent Application No. 62 / 410,317, filed Oct. 19, 2016. By this reference to the patent application, its contents are hereby incorporated herein.

[0002] Field of the Invention

[0002] The present invention relates to an automated staining system and method for biological samples, and more particularly to a system and method for accurately processing cell and tissue samples so as to help conserve both valuable samples and reagents.

Background Art

[0003]

[0003] There are currently three major types of automated staining equipment available, including dip and dunk strainers, puddle strainers, and thin-film stainers. Each of these three types of stainers is used to enable the localization confirmation of cell structures (e.g., nuclei and cell membranes) and / or specific cell components (e.g., protein and nucleic acid markers) and to provide contrast prior to the examination of biological samples (hereinafter "samples") for diagnostic purposes. Typically, a series of reagents are dispensed onto the sample for staining and perhaps for sample preservation purposes. In microscopy, cell samples are typically placed on a substrate such as a microscope slide and processed thereon.

[0004]

[0004] A "dip and dunk" stainer operates by successively immersing a microscope slide or a rack of such slides into a series of reagent volumes (or baths), and is suitable for high-throughput production of slides for examination. Control of the staining process is mainly based on the immersion time and the concentration of the staining reagent in the bath. However, over time, the reagent concentration in the dip and dunk bath changes due to uptake of the reagent by the sample and degradation of the reagent, which is typically left exposed to air in the bath. Additionally, transferring the reagent from one bath to another also contributes to changes in reagent concentration due to dilution and cross-contamination between the reagents. Since it is difficult to control the reagent concentration, automatic dip and dunk stainers are not well-suited for advanced staining protocols, such as immunohistochemistry (IHC) and in situ hybridization (ISH) protocols, where concentration control is crucial for consistency in staining between samples. In addition, antibodies for IHC and nucleic acid probes for ISH protocols are too expensive and precious to be dispensed in bulk in the bath, which is characteristic of dip and dunk stainers. A large amount of waste is generated by dip and dunk stainers, which are not attractive to laboratory personnel who have to handle and dispose of such waste, often in accordance with strict environmental regulations.

[0005]

[0005] The paddle staining technique operates by dispensing sufficient reagent onto a cell sample placed on a horizontally positioned microscope slide, covering the sample with the "paddle" of the reagent, and then leaving it for a predetermined time period for incubation, with or without some effort to mix the reagent, such as by swirling the reagent paddle by injection of compressed air. Once the reagent has contacted the sample for a predetermined amount of time, typically the slide is rinsed to remove the reagent so that fresh reagent can be dispensed. In some cases Therefore, during certain staining protocols, the slides must be washed multiple times to obtain confirmation that the first reagent has been completely removed before dispensing a second potentially incompatible reagent into the sample. The paddle technique has enabled the automation of a wide range of advanced IHC and ISH staining protocols. However, the amount of reagent sufficient to cover a typical tissue sample with a paddle is large, and a portion of the reagent remains unused without reacting with the sample, resulting in waste. Further, considering the amount of rinsing agent, the amount of waste that may be generated by a paddle stainer during a given staining protocol can be very large, and its disposal remains a burden on laboratory staff.

[0006]

[0006] The "thin film" stainer aims to reduce the reagent volume and conserve precious reagents by confining the reagent in the capillary space between the surface of the microscope slide and a second surface such as a cover tile or cover slip. Since the uptake of the reagent by the sample varies by location, depletion of the reagent may occur within the capillary space, leading to a concentration gradient and then inconsistent staining across the sample, potentially making the analysis of the staining pattern uncertain. Although mixing can reduce the staining gradient to some extent by supplying reagent replenishment to the depleted regions, this approach often complicates the design of the stainer.

[0007]

[0007] A more recent approach to conserving precious reagents involves the use of a microfluidic applicator for dispensing the staining reagent onto a small area of the sample. For example, Pepper et.al (Journal of Histology, 34: 3. pp123 - 131, 2011 discloses the use of thermal inkjet printing for histological staining. Another example of a microfluidic applicator is disclosed by Lovchik et al. (15th Int. Conf. on Miniaturized Systems for Chemistry and Life Sciences, Oct. 2 - 6, 2011, pp368 - 370, the content of which is hereby incorporated by reference into this application) by deparaffinization.

[0008]

[0008] PCT Application No. PCT / 2016 / EP058801 discloses a system and method that utilize directed microfluidic reagent dispensing to a sample of a reagent, providing an important step towards the realization of such an integrated system. By citing this patent application herein, its content is hereby incorporated into this application to the extent not inconsistent with this disclosure. Briefly stated, for droplet on - demand application, a main staining composition and a macromolecular reagent composition are provided. Also disclosed is a method of staining a tissue sample by positioning a droplet on - demand print head (e.g., an inkjet print head or other droplet dispensing means) in proximity to a portion of the tissue sample and dispensing a predetermined amount of a staining reagent from the print head to that portion of the tissue sample at a predetermined rate, and this method can be performed multiple times while monitoring the process. For example, by measuring the staining intensity on the sample, if the measured staining intensity does not reach a predetermined threshold value, the dispensing of the reagent can be repeated. The dispensing can be performed with or without using an overlying fluid layer.

Summary of the Invention

Problems to be Solved by the Invention

[0009] ​​Disclosed herein are systems and methods that enable fully automated staining of a wide variety of sample types (e.g., frozen tissue sections, paraffin-embedded tissue sections, hematology and cytology samples) placed on a substrate (such as a microscope slide), preserve tissue morphology, further conserve valuable reagents, and, in certain embodiments, make maximum use of a microfluidic reagent dispenser to conserve valuable reagents and achieve a more complete control over the staining process, which helps extract additional diagnostic information from the sample.

[0010]

[0010] Previous methods have not addressed the dispensing of less valuable bulk reagents (such as washing reagents, buffers, and deparaffinization reagents) that are required to prepare most or all samples for the initial and subsequent dispensing of staining reagents, have not adequately addressed the protection of tissue during processing, and are not considered suitable for automatically preparing samples for encapsulation. In view of this, and as noted above, the present disclosure discloses an integrated method and system that advances the automation of the entire staining process while conserving valuable reagents and reducing waste. Also needed are methods and systems that not only help preserve valuable reagents but also make better use of valuable biological samples in obtaining additional diagnostic information.

Means for Solving the Problems

[0011]

[0011] In one aspect of the present disclosure, the system includes a microfluidic reagent applicator, and It includes a bulk fluid applicator, a fluid aspirator, a sample substrate holder, at least one relative motion system, and a control system. In other embodiments, the system further includes a sample imaging system. In certain embodiments, the bulk fluid applicator and the fluid aspirator are combined into one unit of the system. In more specific embodiments, the microfluidic reagent applicator, the bulk fluid applicator, and the fluid aspirator are combined into one unit of the system. In yet more specific embodiments, the first bulk fluid applicator can be a microfluidic reagent applicator, and in some embodiments, a second bulk fluid applicator is included in the system. Such a second bulk fluid applicator can further be incorporated into one unit of the system together with the fluid applicator. In yet more specific embodiments, when the bulk fluid applicator and the bulk fluid aspirator are incorporated into one unit, the aperture of the bulk fluid applicator and the aperture of the bulk fluid aspirator are separated by at least a distance of 0.1 mm, for example, at least a distance of 0.5 mm such as at least a distance of 1.0 mm.

[0012]

[0012] In another aspect of the present disclosure, the method includes obtaining an image of a sample on a substrate, determining the position of the sample on the substrate, and moving a microfluidic reagent applicator, a bulk fluid applicator, or both to a location on the substrate where the sample is positioned. In one embodiment, the method includes dispensing bulk fluid to a location on the substrate where the sample is positioned and removing bulk fluid from the location on the substrate where the sample is positioned. In certain embodiments, the sample is a paraffin-embedded tissue sample, and the step of determining the position of the sample on the substrate includes detecting a portion of the paraffin section containing the sample and substantially dispensing bulk fluid only to the portion of the paraffin section where the sample is located within the paraffin section. In a more specific embodiment, the bulk fluid includes a deparaffinization reagent. Thus, wells can be formed in the nonpolar paraffin surrounding the sample and used to hold water, aqueous solutions (such as buffers, antibody solutions, or nucleic acid solutions), and other polar reagents (such as humectants) on the sample.

[0013]

[0013] In a more specific embodiment, the sample is a paraffin-embedded tissue or cell sample, and the method further includes selecting two or more distinct portions of the location on the substrate where the sample is located and producing two or more wells in the paraffin located on two or more distinct portions selected at the location where the sample is located by dispensing a deparaffinization fluid to the two or more distinct portions using a bulk fluid applicator. In an even more specific embodiment, the method further includes dispensing the deparaffinization fluid to one selected from two or more distinct portions of the location where the sample is located and simultaneously removing the deparaffinization fluid from the sample using a fluid aspirator. Advantageously, the bulk fluid applicator and the fluid aspirator are 1 and the fluid aspirator are 1 Combined into one unit and moved together to simultaneously dispense and remove the deparaffinizing fluid, thereby quickly removing paraffin from a selected portion of the sample.

[0014]

[0014] In one embodiment, the separation distance between the aperture of the bulk fluid applicator and the aperture of the fluid aspirator is widened to at least 1.0 mm or more (up to 10 mm, up to about 20 mm, up to about 30 mm, or more, e.g., up to about 100 mm, up to about 200 mm, up to about 300 mm, or up to about 1 cm or such greater distances)), and it is possible to maintain a connecting fluid flow between the applicator aperture and the aspirator aperture. In a particular embodiment, the bulk fluid applicator and the fluid aspirator can be a pair of needles separated from each other (e.g., a pair of needles separated by about 1 mm to about 100 mm, about 2 mm to about 50 mm, or about 3 mm to about 10 mm), and a connecting fluid flow can be maintained between the two needles to form a "fluid knife". In this way, for example, the fluid knife can be moved across the entire sample area to selectively deparaffinize all or part of a paraffin-embedded tissue sample. In a more specific embodiment, such a fluid knife can generally be used to create (prepare) a generally square or rectangular well over a selected portion of the sample, and according to a staining protocol further deposit (deposit) and remove other reagents into this well. In an even more specific embodiment, a pair of needles can be rotated about a central axis to form a rotating liquid knife, which can be used to create a circular well over a selected portion of the sample when used to pour out and remove the deparaffinizing fluid. In any case, individual wells can be formed over a selected portion of the sample, and different diagnostic assays can be performed within these separate wells on one sample, thereby obtaining additional diagnostic information from one valuable specimen.

[0015]

[0015] In systems of other aspects of the present disclosure, at least two adjacent microfluidic dispenser ports of one microfluidic reagent dispenser are in fluid communication with at least two separate reagent reservoirs of the microfluidic reagent dispenser. For example, in a matrix of microfluidic dispenser ports of a piezoelectric ink jet printer head or a thermal ink jet printer head, alternate rows or alternate columns of this matrix are in fluid connection with at least two separate reagent reservoirs of the microfluidic reagent dispenser. In an alternative embodiment, alternate microfluidic dispenser ports within one or more rows or columns of a matrix of microfluidic dispenser ports of a piezoelectric ink jet printer head or a thermal ink jet printer head may be in fluid communication with at least two separate reagent reservoirs of the microfluidic dispenser. In certain embodiments, it is also possible to fluidly connect at least two or more different subsections of a matrix of microfluidic dispenser ports of a piezoelectric ink jet printer head or a thermal ink jet printer head to at least two or more separate reagent reservoirs. In other embodiments, particularly when the reagents are compatible with each other (such as primary, secondary, and detection system antibodies and reagents), a valve can control which reagent is delivered sequentially to a matrix of microfluidic dispenser ports according to a given staining protocol.

[0016]

[0016] In a method in other aspects of the present disclosure, at least two staining reagents are deposited sequentially or simultaneously at substantially the same position on a tissue sample such that the at least two staining reagents contact the sample simultaneously. Even reagents that are not normally compatible can be dispensed into the sample simultaneously or in rapid succession from separate microfluidic reagent dispensers (or separate microfluidic dispenser ports of one microfluidic reagent dispenser). For example, hematoxylin and eosin (H&E) can be deposited together on the sample, significantly shortening the time required to prepare an H&E stained sample.

[0017] Another aspect of the present disclosure is a method for automated processing of at least a portion of a sample supported on a substrate. a non-transitory computer-readable medium for performing a method for detecting a sample on a substrate, the memory including: (a) instructions for obtaining an image of the sample on the substrate; (b) instructions for automatically locating the position of the sample on the substrate; and (c) instructions for dispensing a fluid at the position of the sample on the substrate.

[0018] In summary, the disclosed systems and methods represent an improvement to the development, quality, and patient-safety processes involved within the histology tissue staining industry. In one embodiment, a reagent deposition device is provided that ensures that any dispensed reagent does not form a thin boundary layer of fluid. The paddle is configured to penetrate and allow for replenishment of staining reagent in communication with the sample. While not wishing to be bound by any particular theory, it is believed that current staining techniques rely on paddles of staining reagent that passively diffuse a concentration gradient into the tissue sample. In these staining systems, which are believed to lack active mixing of reagent at the paddle-tissue interface, diffusion of stain into the tissue is affected by the build-up of a stain concentration depleted layer at the interface. The present disclosure is believed to provide an improvement over prior art staining techniques by overcoming the limitations of passive dye diffusion by (i) creating a dye film with a thickness that approaches the thickness of the depletion layer, and (ii) replenishing dye molecules at the depletion layer.

[0019]

[0019] Further features and advantages of the disclosed systems and methods will become apparent from the detailed description that follows, when considered in conjunction with the accompanying drawings. [Brief description of the drawings]

[0020]

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DETAILED DESCRIPTION OF THE INVENTION

[0021]

[0046] As used herein, the singular terms "a," "an," and "the" shall be construed to include plural referents unless the context clearly dictates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly dictates otherwise.

[0022]

[0047] The terms "comprising," "including," "having," etc. are used interchangeably and shall have the same meaning. Similarly, the terms "comprises," "includes," "has," etc. are also used interchangeably and shall have the same meaning. Specifically, each of these terms is defined in accordance with the general U.S. patent law definition of "comprising" and is thus construed as an open term meaning "including at least the following" and is further construed not to exclude additional features, limitations, aspects, etc. Thus, for example, "configured 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 involving 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 this specification in a particular order, one of ordinary skill in the art will recognize that the order of steps and processes may be varied unless a particular order is clearly indicated by the context.

[0023]

[0048] As used herein, the term "about" refers to a number to which a reference is made. ±1 to 10% of a value, for example ±1 to 5% of the quoted figure, such as ±1 to 2% of the quoted figure.

[0024]

[0049] As used herein, the term "substantially" means , refers to at least 90%, e.g., refers to at least 95%, such as at least 99% of the objects referred to by this term.

[0025]

[0050] As used herein, the term "antibody" refers to an immunoglobulin or immunoglobulin-like molecule, and by way of example and not limitation, includes IgA, IgD, IgE, IgG, and IgM, combinations thereof, and similar molecules produced during an immune response in any vertebrate (e.g., in mammals such as humans, goats, rabbits, and mice), that specifically bind to a target molecule (or a group of molecules very similar to the target) and substantially exclude binding to other molecules, as well as antibody fragments (e.g., F(ab’)2 fragments, Fab’ fragments, Fab’-SH fragments, and Fab fragments as known in the art), recombinant antibody fragments (e.g., sFv fragments, dsFv fragments, bispecific sFv fragments, bispecific dsFv fragments, F(ab)’2 fragments, single-chain Fv proteins ("scFv"), disulfide-stabilized Fv proteins ("dsFv"), diabodies, and triabodies, and camelid antibodies. An antibody further refers to a polypeptide ligand that specifically recognizes and binds to an epitope of an antigen and includes at least a light chain or a variable region of a heavy chain immunoglobulin. An antibody may be composed of a heavy chain and a light chain, each referred to as a variable heavy chain (VH) region and a variable light chain (VL) region, which have variable regions. The VH region and the VL region together are involved in binding to an antigen recognized by the antibody. The term "antibody" also includes intact immunoglobulins, as well as variants and portions thereof. Specifically recognize and bind to, and refers to a polypeptide ligand that includes at least a light chain or a variable region of a heavy chain immunoglobulin. An antibody may be composed of a heavy chain and a light chain, each referred to as a variable heavy chain (VH) region and a variable light chain (VL) region, which have variable regions. The VH region and the VL region together are involved in binding to an antigen recognized by the antibody. The term "antibody" also includes intact immunoglobulins, as well as variants and portions thereof.

[0026]

[0051] As used herein, the term "antigen" refers to a compound, composition, or substance that can be specifically bound by the product of specific humoral or cellular immunity, such as an antibody molecule Or a T cell receptor. Antigens can be any type of molecule, including, for example, haptens, simple metabolites, sugars (e.g., oligosaccharides), lipids, and hormones, as well as macromolecules such as complex carbohydrates (e.g., polysaccharides), phospholipids, nucleic acids, and proteins.

[0027]

[0052] As used herein, the term "biological sample" or "sample" refers to any solid or fluid sample obtained from, excreted by, or secreted by any living organism, including but not limited to single-celled organisms such as bacteria, yeast, protozoa, and amoeba, and multicellular organisms (including plants or animals, such as healthy or seemingly healthy human subjects, or samples from human patients suffering from a condition or disease to be diagnosed or studied, such as cancer). Specifically, the sample can be suitable for histochemical or cytochemical analysis, such as a sample that retains the morphological characteristics of the cells and / or tissues to be analyzed. For example, a biological sample can be a body fluid obtained from, for example, blood, plasma, serum, urine, bile, ascites, saliva, cerebrospinal fluid, aqueous humor or vitreous humor, or any bodily secretion, effusion, or exudate (e.g., a fluid obtained from an abscess or any other site of infection or inflammation), or a fluid obtained from a joint (e.g., a normal joint or a diseased joint). A biological sample can also be a sample obtained from any organ or tissue (including autopsy specimens such as biopsies or tumor biopsies), or it can include cells (either primary cells or cultured cells), or a medium conditioned by any cell, tissue, or organ. In one example, the biological sample is a nuclear extract. In a particular example, the sample is a quality control sample. In a particular example, the sample is a test sample. For example, a test sample can be a cell, tissue, or cell pellet section prepared from a biological sample obtained from a subject. In one example, the subject is a subject at risk of or suffering from a disease. The sample can be prepared using any method known to those of ordinary skill in the art. The sample is a routine ​For cleaning, it can be obtained from a subject or can also be obtained from a subject suspected of having a disorder such as a genetic abnormality, an infection, or a neoplasm. Also, the embodiments described for the disclosed method can be applied to samples that do not have genetic abnormalities, diseases, disorders, etc., referred to as "normal" samples. The sample can contain a plurality of targets that can specifically bind by one or more detection probes. In a specific example, the sample is a tissue section excised from a paraffin-embedded tissue block placed (and perhaps baked thereon) on a microscope slide. In another specific example, the sample is a cytological or hematological sample prepared by depositing cells on a microscope slide (by contacting a filter on which the cells are collected to form a smear or by printing the cells in a pattern across the surface of the microscope slide).

[0028]

[0053] As used herein, "drop-on-demand", "droplet-on-demand", or "droplet-based" (and other similar terms or phrases) refers to a staining technique that deposits discrete droplets of a reagent onto a target sample, as opposed to "flooding" the slide or the sample thereon with the reagent. In certain embodiments, the droplet-on-demand technique utilizes inkjet technology or piezoelectric technology. In some embodiments disclosed herein, the droplet dispensing technique is facilitated by using an inkjet print head or similar technology.

[0029]

[0054] As used herein, the term "humectant" refers to a substance 、For example, it refers to a hygroscopic substance used to moisten a tissue sample, which is the opposite of a desiccant. This is often a molecule containing various hydrophilic groups, most often hydroxyl groups, but amines and carboxyl groups, and sometimes esterified ones, may also be encountered (the affinity to form hydrogen bonds with water molecules is an essential property). A water retention agent is thought to attract moisture in the nearby air by absorption and hold it, and draw water vapor into and / or under the surface of the organism / subject. In contrast, a desiccant also attracts ambient moisture, but adsorbs it rather than absorbs it by condensing water vapor on the surface as a coating layer. In the context of inkjet deposition or similar techniques, it is most reasonable that a water retention agent is important for maintaining a sustainable nozzle. In certain embodiments, it is important to hydrate a tissue sample or a biological sample during thin film processing.

[0030]

[0055] The term "inkjet" as used in this disclosure refers to a group of drop-on-demand technologies that use piezoelectric (or thermal) elements to actuate droplets from a dispensing manifold. This may include direct and non-contact methods common in the commercial printing industry, or those used outside the commercial printing industry.

[0031]

[0056] As used herein, the term "immunohistochemistry" refers to a method of determining the presence or distribution of an antigen in a sample by detecting the interaction of the antigen with a specific binding agent such as an antibody. The sample is contacted with the antibody under conditions that allow antibody-antigen binding. Antibody-antigen binding is detectable by a detectable label conjugated to the antibody (direct detection), or by a detectable label conjugated to a secondary antibody that specifically binds to the primary antibody (indirect detection).

[0032]

[0057] As used herein, the term "primary antibody" refers to an antibody that specifically binds to a target protein antigen in a tissue sample. A primary antibody is generally the first antibody used in immunohistochemistry. Primary antibodies also include antibodies conjugated to another molecule (e.g., a label, a hapten, etc.). A primary antibody can also serve as a "detection probe" to detect a target in a tissue sample.

[0033]

[0058] As used herein, the term "primary stain" A dye or similar molecule that enhances contrast in tissue samples. In an embodiment, the primary stain is one that directly "labels" biological structures in or on cells without the use of specific binding agents such as antibodies. Some examples of primary stains include hematoxylin and eosin. Other examples of primary stains include acridine orange, bismarck brown, carmine, Coomassie blue, cresyl violet, crystal violet, DAPI ("2-(4-amidinophenyl)-1H-indole-6-carboxamidine"), ethidium bromide, acid fuchsin, Hoechst stain (bis-benzimidazole derivatives Hoechst 33342 and Hoechst 33258), iodine, malachite green, methyl green, methylene blue, neutral red, Nile blue, Nile red, osmium tetroxide, rhodamine, and safranine. Other examples of primary stains include stains used to stain bacteria (Gram-positive or Gram-negative stains), stains used to identify endospores (endospore stains), and stains used to aid in the identification of species of Mycobacterium tuberculosis. Stains used for this purpose (Ziehl-Neelsen stain), Papanicolaou staining kits (using a combination of hematoxylin, orange G, eosin Y, light green SF yellowish, and sometimes Bismarck brown Y), periodic acid Schiff stain ("PAS stain"), silver stain, etc. are included. Further non-limiting primary stains include (i) histological stains for selectively indicating Mycobacterium and other acid-fast organisms or components (e.g., AFB III staining kit available from Ventana Medical Systems Inc. (hereinafter Ventana, Tucson, Arizona, United States of America)), (ii) histological stains for differentiating acidic mucin from neutral polysaccharides e.g., Alcian blue for PAS, also available from Ventana), (iii) histological stains for indicating weakly acidic mucopolysaccharides (e.g., Alcian blue staining kit, also available from Ventana), (iv) histological stains for Helicobacter pylori ​(For example, an Alcian Yellow staining kit similarly available from Ventana), (v) histological stains for selectively indicating amyloid (e.g., a Congo Red staining kit similarly available from Ventana), (vi) histological stains for distinguishing acidic mucin from neutral polysaccharides (e.g., a diastase kit similarly available from Ventana), (vii) histological stains for indicating elastic fibers in tissue sections (e.g., an elastic staining kit similarly available from Ventana), (viii) histological stains for distinguishing white blood cells in bone marrow and other hematopoietic tissues (lymph nodes) (e.g., a Giemsa staining kit similarly available from Ventana), (ix) histological stains for indicating polysaccharides in the cell walls of fungi and other opportunistic infectious organisms, including but not limited to pathogenic fungi such as Aspergillus and Blastomyces, and other opportunistic infectious organisms such as Pneumocystis carinii, which can distinguish them (e.g., a GMS II staining kit similarly available from Ventana), (x) histological stains for indicating Gram-negative and Gram-positive bacteria (e.g., a Gram staining kit similarly available from Ventana), (xi) histological stains used to study connective tissue, muscle, and collagen fibers (e.g., Green for Trichrome similarly available from Ventana), (xii) histological stains for detecting iron pigments in bone marrow, hemochromatosis tissue, and hemosiderosis (e.g., an iron staining kit similarly available from Ventana), (xiii) histological stains for indicating capillary basement membranes (e.g., either a Jones H&E staining kit or Jones Light Green staining kit, both similarly available from Ventana), (xiv) histological stains for fungal detection (e.g., Light Green for PAS similarly available from Ventana), (xv) histological stains for detecting acidic mucopolysaccharides (mucin) (e.g., a Muciarmine staining kit similarly available from Ventana), (xvi) positive reticular fibers, basement membranes, fungi 、and histological stains used to indicate the presence of glycogen, including stains that can assist in the identification of neutral mucopolysaccharides, or stains that can assist in differentiating PAS-positive secretory adenocarcinoma from undifferentiated PAS-negative squamous cell carcinoma (e.g., PAS staining kits similarly available from Ventana), (xvii) histological stains for demonstrating reticular fibers (e.g., Reticulin II staining kits similarly available from Ventana), (xviii) histological stains used to study certain argyrophilic microorganisms (e.g., Steiner II staining kits similarly available from Ventana), (xix) histological silver stains for assisting in the identification of causative organisms of certain diseases such as certain gastric ulcers (H. pylori), Lyme disease, Legionnaires' disease, cat scratch fever, etc. (e.g., Steiner staining kits similarly available from Ventana), (xx) histological stains for studying connective tissue, muscle, and collagen fibers (e.g., Trichrome II Blue staining kits similarly available from Ventana), (xxi) histological stains for studying connective tissue, muscle, and collagen fibers (e.g., Trichrome staining kits, Trichrome III Blue staining kits, or Trichrome III Green staining kits, respectively, similarly available from Ventana) are included. Also, one of ordinary skill in the art will recognize that there are other primary stains or dyes that can be used in combination with the kits, methods, and compositions of the present disclosure (e.g., primary staining compositions, reagent compositions).

[0034]

[0059] As used herein, the term "reagent" refers to morphological It may also refer to any fluid deposited on a tissue section or cytological sample, used in connection with (e.g., hematoxylin and eosin), immunohistochemical, or special staining. This includes oils, organic substances, and cross-linking reagents for removing wax (i.e., dewaxing), washes, rinses, diluents, or buffers used to set reaction conditions, dilution reagents for bringing to the appropriate concentration, stopping the reaction, or washing away excess reactants, small molecule dyes used in morphological and special staining, antibodies, antibody conjugates, enzymes, multimers, amplifiers, chromogenic substrates, fluorescent detection chemicals, chemiluminescent substrates, and enzyme reaction cofactors, but is not limited thereto.

[0035]

[0060] As used herein, a "surfactant" is classified as anionic, cationic, or nonionic depending on its mode of chemical action. Generally, a surfactant reduces the interfacial tension between two liquids. Surfactant molecules typically have a polar or ionic "head" and a nonpolar hydrocarbon "tail". Upon dissolution in water, surfactant molecules aggregate and form micelles, in which the nonpolar tails face inward and the polar or ionic heads face outward toward the aqueous environment. The nonpolar tails create a nonpolar "pocket" within the micelle. Nonpolar compounds in solution are sequestered within the pockets formed by surfactant molecules, thus allowing the nonpolar compounds to remain mixed within the aqueous solution. In certain embodiments, the use of a surfactant can result in uniform diffusion of reagents across a tissue section and can also reduce background staining.

[0036]

[0061] As used herein, "target" can be a specific tissue in a biological sample or a specific molecule or marker in a biological sample. Examples of targets include antigens (including haptens), antibodies, and enzymes. Still other examples of targets typically include proteins, peptides, nucleic acids, sugars, and lipids. A reagent for use in the present disclosure may be one capable of converting a target substance present in a biological sample into a detectable form (such as visually) so that the localization of the target can be detected.

[0037]

[0062] One aspect of the present disclosure is an automated biological sample staining system that includes at least one microfluidic reagent applicator, at least one bulk fluid applicator, at least one fluid aspirator, and at least one sample substrate holder. In certain embodiments, the automated biological sample staining system further includes at least one relative motion system for moving one or more of the sample substrate holder(s), microfluidic reagent applicator(s), bulk fluid applicator(s), and fluid aspirator(s) together or separately in any combination. In certain embodiments, the automated biological sample staining system further includes a control system programmed to perform at least one staining protocol on a sample placed on a substrate held within the sample substrate holder. In certain embodiments, the system includes more than one microfluidic reagent applicator.

[0038]

[0063] In one embodiment, the control system controls one or more of at least one microfluidic reagent applicator, at least one bulk fluid applicator, at least one fluid aspirator, at least one sample substrate holder, and at least one relative motion system to perform the individual steps of at least one staining protocol. In one embodiment, the control system can further control one or more auxiliary subsystems that operate in combination with the above components to treat a sample according to a specific staining protocol. The number of staining protocols stored in the memory of the control system (e.g., non-transitory memory), as well as the number of instructions and parameters measured and / or applied to perform the individual steps of a specific staining protocol, is not limited and, conversely, is typically scaled according to the complexity of the overall system. That is, in an embodiment for individual slide staining according to one specific staining protocol (such as a small-scale system for rapid H&E staining of frozen tissue sections in the operating room), the number of instructions and parameters stored in the control system memory can be minimized. However, in a complex system that receives various different types of samples to be treated according to a large number of staining protocols on separate substrate holders, the number of protocols, instructions, stored parameters, measured parameters, processing algorithms, etc. can be as large as necessary to reliably and repeatedly execute any number of staining protocols.

[0039]

[0064] Examples of additional subsystems that can communicate with and be controlled by a control system and / or be moved by at least one relative motion system include one or more of at least one sample imaging system, at least one air knife, at least one waste management system, and at least one sample identification system. Other examples of additional subsystems include one or more reagent storage units (which can be cooled), one or more reagent transfer systems, and one or more substrate transfer systems. Still other examples of additional subsystems that can communicate with and be under the control of a control system will be described below with reference to FIG. 1.

[0040]

[0065] In other embodiments, at least one fluid aspirator is replaced with at least one air knife. An air knife is used to facilitate the movement of fluid from a sample using a compressed gas such as compressed air or nitrogen. For example, instead of sucking fluids from a sample and directing them towards waste, the fluids can be moved to a waste container or a waste receiver leading to the waste container, or "blown off" into it.

[0041]

[0066] In certain embodiments, various system components (e.g., those identified in FIG. 1) may be combined to form one or more reagent management units. Each reagent management unit may have the same or different configurations (e.g., configuration means the type of system component, the number of system components, or the quality of any one system component). In view of this, the systems disclosed herein may comprise one or more reagent management units, e.g., one or more reagent management units, two or more reagent management units, three or more reagent management units, or four or more reagent management units.

[0042]

[0067] In one embodiment, at least one bulk fluid applicator and at least one fluid aspirator are combined in at least one first type of reagent management unit. In other specific embodiments, at least one microfluidic reagent applicator, at least one bulk fluid applicator, and at least one fluid aspirator are combined in at least one second type of reagent management unit. In other specific embodiments, at least one microfluidic reagent applicator, at least one bulk fluid applicator, and at least one air knife are combined in at least one third type of reagent management unit. In yet another specific embodiment, at least one microfluidic reagent applicator, at least one bulk fluid applicator, at least one fluid aspirator, and at least one air knife are combined in at least one fourth type of reagent management unit. Depending on the system configuration, the system can include any combination of two or more of the first type, second type, third type, and fourth type of reagent management units. In one embodiment, the reagent management unit can include, for example, pumps, reservoirs, valves, etc., and can further include a controller for controlling these to deliver a predetermined amount of a predetermined fluid. In one embodiment, the reagent management unit can further include other means for delivering one or more reagents, which can be solid or liquid. For example, the reagent management unit can also include a reconstitution unit for dissolving a solid reagent in a liquid for dispensing into a sample. Alternatively, the reagent management unit can further include one or more single-dose reagent applicators, such as a blister pack and associated mechanisms for managing the contents of the blister and dispensing them into contact with the sample.

[0043]

[0068] In one embodiment, at least one bulk fluid applicator and at least one fluid aspirator comprise a pair of needles. In yet another specific example, the pair of needles are separated by at least a distance of 0.5 mm, such as at least a distance of 0.1 mm, for example at least a distance of 1.0 mm.

[0044]

[0069] Regardless of the type of at least one reagent management unit (i.e., the selection and / or number of system components), the at least one reagent management unit can be coupled to at least one relative motion system, or at least one sample substrate holder can be coupled to at least one relative motion system, or both the at least one reagent treatment unit and the at least one sample substrate holder are coupled to at least one relative motion system. Depending on the system configuration, the system can include any combination of couplings between different types of reagent management units, different sample substrate holders, and different relative motion systems, and relative motion can occur between each component.

[0045]

[0070] In one embodiment, the distance between the aperture of the bulk fluid applicator and the aperture of the bulk fluid aspirator can be at least 1.0 mm or more (up to about 10 mm, up to about 20 mm, up to about 30 mm, or more, for example up to about 1 cm, such as up to about 100 mm, up to about 200 mm, up to about 300 mm, or more), and still maintain a fluid flow connection between the applicator aperture and the aspirator aperture. In certain embodiments as described above, the bulk fluid applicator and the bulk fluid aspirator can be a pair of needles separated from each other (e.g., by only about 1 mm to about 100 mm, such as from about 2 mm to about 50 mm, or from about 3 mm to about 10 mm), and a connecting fluid flow can be maintained between the two needles to form a "fluid knife".

[0046]

[0071] For example, in one embodiment, to selectively deparaffinize all or a portion of a paraffin-embedded tissue sample, a fluid knife can be moved across the entire sample area by relative motion. In a more specific embodiment, such a fluid knife can be used to create a square or rectangular well over a selected portion of the sample, into which other reagents can be deposited or removed according to a staining protocol. In an even more specific embodiment, a pair of needles can be rotated about a central axis to form a rotating liquid knife, which can be used to create a circular well over a selected portion of the sample when used to dispense and remove the deparaffinizing fluid. In a particular embodiment of the disclosed system, at least one microfluidic reagent applicator comprises at least one micro-fabricated chip applicator as disclosed in Lovchik et al. (15th Int. Conf. on Miniaturized Systems for Chemistry and Life Science, Oct. 2 - 6, 2011, pp. 368 - 370), the contents of which are hereby incorporated by reference. In other particular embodiments, at least one microfluidic reagent applicator comprises a droplet-on-demand actuator, which can be, for example, a piezoelectric actuator or a thermal actuator. In a more specific embodiment, the disclosed system can include any combination of two or more of a micro-fabricated chip applicator, a piezoelectric applicator, and a thermal applicator. For example, a piezoelectric actuator is susceptible to degradation by sheer force or thermally unstable reagents

[0047]

[0072] In a particular embodiment of the disclosed system, at least one microfluidic reagent applicator comprises at least one micro-fabricated chip applicator as disclosed in Lovchik et al. (15th Int. Conf. on Miniaturized Systems for Chemistry and Life Science, Oct. 2~6, 201, pp368~370). The contents of this document are hereby incorporated by reference. In other particular embodiments, at least one microfluidic reagent applicator comprises a droplet-on-demand actuator, which can be, for example, a piezoelectric actuator or a thermal actuator. In a more specific embodiment, the disclosed system can include any combination of two or more of a micro-fabricated chip applicator, a piezoelectric applicator, and a thermal applicator. For example, a piezoelectric actuator is susceptible to degradation by sheer force or thermally unstable reagents In a particular embodiment of the disclosed system, at least one microfluidic reagent applicator comprises at least one micro-fabricated chip applicator as disclosed in Lovchik et al. (15th Int. Conf. on Miniaturized Systems for Chemistry and Life Science, Oct. 2 - 6, 2011, pp. 368 - 370), the contents of which are hereby incorporated by reference. In other particular embodiments, at least one microfluidic reagent applicator comprises a droplet-on-demand actuator, which can be, for example, a piezoelectric actuator or a thermal actuator. In a more specific embodiment, the disclosed system can include any combination of two or more of a micro-fabricated chip applicator, a piezoelectric applicator, and a thermal applicator. For example, a piezoelectric actuator is susceptible to degradation by sheer force or thermally unstable reagents It may be well selected for dispensing a fluid containing a drug, while a microfabricated chip applicator or a thermal actuator is a reagent that is not easily deteriorated by sheer force Or for dispensing a fluid containing a reagent that is not thermally unstable, respectively, it may be well selected. The deterioration of a specific reagent can be determined by comparing its staining performance on the paddle or by a thin film staining system as described in the previous background.

[0048]

[0073] In other specific embodiments, the microfluidic reagent applicator comprises an integrated reagent reservoir, such as one that is fluidly connected to a droplet-on-demand actuator. In one embodiment, the microfluidic reagent applicator comprises a remote reagent reservoir, such as a remote reagent reservoir that is fluidly connected to a droplet-on-demand actuator. In still other specific embodiments, the microfluidic reagent applicator can comprise a replaceable reservoir. When combined with a droplet-on-demand actuator and a replaceable reagent reservoir, this replaceable reservoir is fluidly connected to the microfluidic reagent applicator upon the combination of these two. Also, the microfluidic reagent applicator can comprise an intermediate reagent reservoir. The intermediate reagent reservoir is fluidly connected to the remote reagent reservoir and is supplied by the remote reagent reservoir. For example, in a more specific embodiment, the micro reagent applicator comprises a droplet-on-demand actuator integrated with an intermediate reagent reservoir that is fluidly connected to the remote reagent reservoir.

[0049]

[0074] Those skilled in the art will appreciate that the disclosed system can include any combination of reservoir configurations and / or any combination of bulk fluid applicators as described herein. For example, valuable primary antibodies and nucleic acid probes can be supplied in a microfluidic applicator that includes an integrated reservoir, which is moved in and out of a cooled reagent storage system and transported to a particular sample substrate holder when required in a particular staining protocol. Alternatively , such valuable reagents can be held in an exchangeable reservoir, which is moved in and out of a cooled reagent storage system and coupled to a microfluidic reagent applicator such as a droplet-on-demand actuator. On the other hand, less valuable reagents such as bulk fluids containing wash solutions, deparaffinizing fluids, etc., can be held in a remote reagent reservoir that can be easily refilled and fluidly connected to a microfluidic reagent applicator or a bulk fluid applicator (such as through tubing and perhaps via an intermediate reservoir). Similarly, reagents used in multiple staining protocols (e.g., detection reagents such as secondary antibodies, tertiary antibodies, antibodies conjugated to enzymes, enzyme substrates, etc.) can be held in a reservoir proximate to one or more sample substrate holders. These reservoirs are used to perform detection chemistries and are fluidly connected to one or more microfluidic reagent applicators either directly or through tubing (and perhaps via an intermediate reservoir). In certain embodiments, all of the different reagents used for a particular detection chemistry (such as di-aminobenzidine detection of primary antibody binding, etc.) are held in a reservoir fluidly connected to one microfluidic reagent applicator (either directly, or through tubing, or through tubing and intermediate reservoirs) and are directed onto a sample, sequentially or simultaneously, in any combination, through one microfluidic reagent applicator. In a more specific embodiment, at least two adjacent microfluidic dispenser ports of one microfluidic reagent dispenser are fluidly connected to at least two separate reagent reservoirs of the microfluidic reagent dispenser. As already explained, for example, in a matrix of microfluidic dispenser ports of a piezoelectric ink jet printer head or a thermal ink jet printer head, alternate rows or alternate columns of this matrix are fluidly connected to at least two separate reagent reservoirs of the microfluidic reagent dispenser. In other alternative embodiments, alternate microfluidic dispenser ports within one or more rows or columns of a matrix of microfluidic dispenser ports of a piezoelectric ink jet printer head or a thermal ink jet printer head are fluidly connected to at least two separate reagent reservoirs of the microfluidic dispenser. In one embodiment, at least two or more different subsections of a matrix of microfluidic dispenser ports of a piezoelectric ink jet printer head or a thermal ink jet printer head are fluidly connected to at least two or more separate reagent reservoirs. In yet another embodiment, particularly when the reagents are compatible with each other (such as in the case of primary, secondary, and detection system antibodies and reagents), which reagent is continuously delivered to the matrix of microfluidic dispenser ports according to a given staining protocol can be controlled by a valve.

[0050]

[0075] Also disclosed herein is a method for automatically treating at least a part of a sample held on a substrate. This method includes the steps of obtaining an image of the sample on the substrate, automatically determining the position of the sample on the substrate, and dispensing a fluid at the position of the sample on the substrate. In one embodiment, the step of dispensing a fluid at the position of the sample on the substrate includes the step of dispensing the fluid substantially only at the position of the sample on the substrate. In a particular embodiment, this method can further include the step of removing the fluid from the part of the substrate where the sample is located. In a more particular embodiment, the sample includes a paraffin-embedded sample, and the step of determining the position of the sample on the substrate includes the step of automatically detecting a part of the paraffin section containing the sample. In an even more particular embodiment, the fluid includes a deparaffinizing fluid, and the step of dispensing the fluid includes dispensing the deparaffinizing fluid onto at least one sub-portion of the paraffin section containing a small part of the sample, and can further include the step of removing the deparaffinizing fluid from at least one sub-portion of the paraffin section containing a small part of the sample to form a well in the paraffin around the small part of the sample. Alternatively, the fluid includes a deparaffinizing fluid, and the step of dispensing the fluid includes dispensing the deparaffinizing fluid onto substantially the portion of the paraffin section containing the sample, and further removing the deparaffinizing liquid from substantially the portion of the paraffin section containing the sample and leaving paraffin on the substrate around the sample to form a well in the paraffin substantially surrounding the sample. In some embodiments, the dispensing of the deparaffinizing fluid and the removal of the deparaffinizing fluid may be performed simultaneously.

[0051]

[0076] ​​Once a well (or wells) is formed around a sample (or one or more small portions of a sample), the method can further include the step of dispensing at least one second fluid into the well (or wells) in the paraffin. Without wishing to be bound by any particular theory, it is believed that the well in the paraffin can trap a polar (such as aqueous) solution because the hydrophobic paraffin forms a barrier to the migration of the polar solution. For example, the second fluid can be one or more of water, buffer solution, antibody solution, dye solution, nucleic acid solution, solvent, surfactant, and water retention agent that enter the well in the paraffin.

[0052]

[0077] In more specific embodiments, the disclosed method includes selecting small portions of two or more distinct samples from the location of a sample on a substrate, and dispensing a deparaffinizing fluid onto the two or more distinct selected small portions of the sample to dissolve paraffin thereon. In certain embodiments, the method can further include removing the deparaffinizing fluid from two or more distinct small portions of the sample to form two or more distinct wells in the paraffin surrounding the two or more distinct small portions of the sample. In even more specific embodiments, the disclosed method includes selecting two or more distinct small portions from an image of consecutive H&E-stained sections of the same sample block from which the sample was obtained. The H&E-stained sections of the consecutive sections (which means sections sliced by a microtome or other means in one of several samples from which two or more distinct small portions were selected) have generally the same overall shape as the sample from which the two or more distinct small portions were selected, so that specific morphological features identified in the image of the consecutive H&E-stained sections can be identified, mapped (by image analysis techniques well known in the art) to similar small portions of the sample, and used to guide further treatment of the sample. For example, two or more distinct small portions of the sample mapped from the consecutive sections are selected to correspond to different morphological features of the sample. In certain embodiments, two or more distinct small portions of the sample mapped from consecutive H&E sections are selected to provide at least one of a positive control or a negative control, and to provide at least one small portion of the sample for comparison with at least one of the positive control and the negative control.

[0053]

[0078] In other embodiments, a method is disclosed for sequentially or simultaneously depositing at least two staining reagents at substantially the same location on a tissue sample. According to embodiments of the present disclosure, even reagents that are considered incompatible with each other, such as hematoxylin and eosin, can be deposited together on the sample from one or more microfluidic reagent dispensers, for example, from adjacent microfluidic dispenser ports or separate microfluidic reagent dispensers, which has the advantage.

[0054]

[0079] In certain embodiments, real-time dispense volume measurement data may be stored and correlated with an identifier of the slide specimen, and the identifier of each dispenser may be associated (affiliate) with the delivery of the reagent to the specimen. This metadata serves purposes of tracking and reporting in histological research and may be stored on the instrument or a host computer. The dispense volume metadata can track the entire slide staining process history. Additionally, continuous performance tracking can be collated for each dispenser identifier over its lifespan. For a given "poor dispense", the "failed" dispenser and the affected specimens can be flagged, for example by software and reported to the histologist by various electronic means (i.e., LED indicator, run report, etc.), enhancing patient safety. The dispense volume metadata may be collected in an external data bank for research and development purposes and this data can be used to qualify and sort new staining kits or individual staining products. Additionally, newly developed reagents may vary in performance over time and may further affect the dispense delivery of the reagent to the specimen slide (i.e., material compatibility between the reagent and the dispenser), so dispense verification tracking can also be used with these newly developed reagents.

[0055]

[0055]

[0080] In one embodiment, a reagent, or a composition containing the reagent, is dispensed from a microfluidic reagent dispenser at a sufficient rate through an immiscible fluid, and the reagent droplets are fed through a thin film of a tissue-preserving fluid medium. Examples of thin film fluids include, but are not limited to, draksol, lymphar, mineral oil, or silicone oil. Generally, preferred attributes include being in a liquid state at room temperature (e.g., 20 - 30 degrees Celsius), having a low surface tension, and having a low vapor pressure. In one embodiment, an immiscible barrier layer is thought to enable the resupply of an aqueous liquid through this barrier to the sample surface. The low surface tension allows the barrier to be coated on the sample as a relatively thin film (height of about 100 μm or less). Furthermore, the low vapor pressure is thought to ensure that the barrier layer evaporates slowly from the sample. By this, it is thought that the reagent is fed into a layer communicating with the sample under the immiscible fluid. In this embodiment, the kinetic energy of the droplet (the product of the mass of the droplet and the impact velocity when the droplet collides with the film) must be greater than the surface tension / energy of the protective layer (and in addition, sufficient additional energy corresponding to the displaced fluid must be supplied), for example, greater than about 9.52x10 J. In one embodiment, the kinetic energy is about 6.23x10 J. Furthermore, to ensure that droplet breakup does not occur upon impact, the Weber number of the droplet must be less than about 18. In one embodiment, the droplet must have a higher density than the protective film to ensure that once the surface is broken, the droplet continues to make direct contact with the sample through the protective layer. It is not limited thereto. Generally, preferred attributes include being in a liquid state at room temperature (e.g., 20 - 30 degrees Celsius), having a low surface tension, and having a low vapor pressure. In one embodiment, an immiscible barrier layer is thought to enable the resupply of an aqueous liquid through this barrier to the sample surface. The low surface tension allows the barrier to be coated on the sample as a relatively thin film (height of about 1 00 μm or less). Furthermore, the low vapor pressure is thought to ensure that the barrier layer evaporates slowly from the sample. By this, it is thought that the reagent is fed into a layer communicating with the sample under the immiscible fluid. In this embodiment, the kinetic energy of the droplet (the product of the mass of the droplet and the impact velocity when the droplet collides with the film) must be greater than the surface tension / energy of the protective layer (and in addition, sufficient additional energy corresponding to the displaced fluid must be supplied), for example, greater than about 9.52x10 J. In one embodiment, the kinetic energy is about 6.23x10 -10 J. In one embodiment, the kinetic energy is about 6.23x10 -10 J. Furthermore, to ensure that droplet breakup does not occur upon impact, the Weber number of the droplet must be less than about 18. In one embodiment, to ensure that the droplet continues to make direct contact with the sample through the protective layer once the surface is broken, the droplet must have a higher density than the protective film.

[0056]

[0081] In other embodiments, the reagent is dispensed into a pre-existing fluid "layer" at a rate sufficient to feed reagent droplets into a thin film that locally conveys the stain through the layer to the fluid-tissue stain depletion layer. This is thought to facilitate replenishment of the reagent at the interface contact point in communication with the sample. On the other hand, this is thought to eliminate the stain depletion boundary layer and, in some cases, improve the staining reaction kinetics that are affected by the diffusion of the staining reagent across the depletion layer. Indeed, for macromolecules such as antibodies, the binding of the molecule to the target is a function of time and concentration. It is thought that dispensing by the devices (and attendant unique mixing) disclosed herein can enhance the effective concentration at the tissue surface and increase the uptake rate by continuously disrupting the thin film with additional reagent material. In this embodiment, the rate is generally in the range of about 5 m / s to about 15 m / s.

[0057]

[0082] An embodiment of an automated biological staining system disclosed in accordance with an embodiment of the present disclosure is shown in FIG. 1. System 100 includes a control system 102. Control system 102 can communicate with various subsystems and communicate with network 104. Network 104 can further communicate with additional automated biological staining systems, pathology laboratory workflow control and tracking systems, and control systems of laboratory information systems (LIS) and / or hospital information systems (HIS). An order for a particular sample prepared in a pathology laboratory can be sent to control system 102 and stored in the memory (not shown) of the control system until the sample reaches system 1 00. For example, a particular sample placed on a microscope slide can be directed towards system 100 by a substrate transfer / substrate movement system 106 (which can be a 1-D or 2-D transfer system such as a conveyor belt or magnetic transfer system). When the sample reaches system 100 (either manually or automatically), a substrate identification system 108 identifies an identifier (such as a unique sample identifier etc.) associated with the microscope slide (e.g., a bar code Based on a label, numerical identifier, or RFID tag (etc.), the sample is identified, and the control system 102 associates this specific sample with a command that designates a processing step (which can be stored in the memory of the control system 102 or sent to the control system through the network 104 along with the command). The image acquisition system 110 can also function as part of the substrate identification system 108, image a sample (such as a paraffin-embedded tissue sample), and generate a map of the sample. This map can be used to process the sample according to a specific protocol. The image can be displayed, for example, on the GUI 114, and the user can interact with the control system 102 (such as via a touch screen) to control the movement and / or processing steps. Also, the GUI 114 can be used, for example, to monitor the progress of the samples processed by the system 100, display warnings, and perform quality control checks.

[0058]

[0083] The control system 102 can include known components such as a processor, an operating system, a system memory, a memory storage device, an input / output controller, input / output devices, and a display device. It may also include a cache memory, a data backup unit, and many other devices. Examples of input devices include a keyboard, a cursor control device (e.g., a mouse), a microphone, a scanner, etc. Examples of output devices include a display device (e.g., a monitor or a projector such as the GUI 114), a speaker, a printer, a network card, etc. The display device may include a display device that provides visual information, which can typically be organized logically and / or physically as an array of pixels. An interface controller can also be included, and the interface controller can be equipped with any of various known software programs or future software programs for providing input and output interfaces. Typically, the interface can be used to receive user input using selection or input means known to those skilled in the relevant art. The interface may also be a touch screen device. In the same or alternative embodiments, an application on a computer may employ an interface that includes what is often called a "command line interface" (CLI). The CLI typically provides text-based interactive processing between the application and the user. Typically, the command line interface presents output through a display device and accepts input as lines of text. For example, in some embodiments, it may include what is called a "shell" such as Unix Shells known to those skilled in the relevant art, or Microsoft Windows Powershell that employs an object-oriented programming architecture such as the Microsoft.NET framework. ​

[0059]

[0084] It will be appreciated by those skilled in the relevant art that the interface may also include one or more GUIs, CLIs, or combinations thereof. The processor may include commercially available processors such as Celeron, Core, or Pentium processors manufactured by Intel Corporation, SPARC processors manufactured by Sun Microsystems, Athlon, Sempron, Phenom, or Opteron processors manufactured by AMD Corporation, or may be one of other processors currently available or to become available in the future. Embodiments of the processor may include what is referred to as a multi-core processor and / or may include those that enable the adoption of parallel processing techniques in a single or multi-core configuration. For example, a multi-core architecture typically includes two or more processor "execution cores". In this example, each execution core can execute as an independent processor enabling parallel execution of multiple threads. Additionally, it will be appreciated by those skilled in the relevant art that the processor may be configured with what is generally referred to as a 32 or 64-bit architecture, or with other architectural configurations currently known or that may be developed in the future. m processors, SPARC processors manufactured by Sun Microsystems, A MD Corporation's Athlon, Sempron, Phenom , or an Opteron processor, or may be one of other processors currently available or to become available in the future. Embodiments of the processor may include what is referred to as a multi-core processor and / or may include those that enable the adoption of parallel processing techniques in a single or multi-core configuration. For example, a multi-core architecture typically includes two or more processor "execution cores". In this example, each execution core can execute as an independent processor enabling parallel execution of multiple threads. Additionally, the processor may be configured with what is generally referred to as a 32 or 64-bit architecture, or with other architectural configurations currently known or that may be developed in the future, which will be appreciated by those skilled in the relevant art.

[0060]

[0085] The processor typically executes an operating system. For example, the operating system may be a Windows-type operating system from Microsoft Corporation, a Mac OSX operating system from Apple Computer Corp., a Unix operating system available from many vendors dows-type operating system, a Mac OSX operating system from Apple Computer Corp., a Unix operating system available from many vendors Or a Linux (registered trademark) - type operating system, or something called open - source, other or future operating systems, or any combination thereof may be used. The operating system interfaces with firmware and hardware in a well - known manner and assists the processor when coordinating and executing the functions of various computer programs that can be written in various programming languages. The operating system typically works in cooperation with the processor to coordinate and execute the functions of other components of the computer. Also, the operating system provides, according to all known techniques, scheduling, input / output control, file and data management, memory management, and communication control, as well as related services.

[0061]

[0086] The system memory can be used to store desired information and can include any of a variety of known memory storage devices or future memory storage devices that can be accessed by a computer. The computer-readable storage medium can include volatile and non-volatile, removable and non-removable media and is implemented in any method or technology for storage of information such as computer-readable instructions, data structures, program modules, or other data. Examples thereof include any commonly available random access memory (RAM), read only memory (ROM), electronically erasable programmable read only memory (EEPROM), digital versatile disk (DVD), magnetic media such as an internal hard disk or tape, optical media such as a read and write compact disk, or other memory storage devices. The memory storage devices can include any of a variety of known devices or future devices and include compact disk drives, tape drives, removable hard disk drives, USB or flash drives, or floppy disk drives. Such types of memory storage devices typically read from and / or write to program storage media such as compact disks, magnetic tapes, removable hard disks, USB or flash drives, or floppy disks. Any of these program storage media, or others currently in use, or those that may be developed in the future, may be regarded as computer program products. As will be appreciated, these program storage media typically store computer software programs and / or data. The computer software programs, also called computer control logic, are typically stored in the system memory and / or in a program storage device associated with the memory storage device for use therewith.In one embodiment, a computer program product is described that comprises a computer-usable medium having control logic (computer software program including program code) stored therein. When executed by a processor, the control logic causes the processor to perform the functions described herein. In other embodiments, depending on the functionality, some implementations may be primarily implemented in hardware, such as using a hardware state machine. Implementing a hardware state machine to perform the functions described herein will be apparent to those skilled in the relevant art. The input / output controller receives information from a user, whether human or machine, local or remote. It can include any of a variety of known devices for receiving and processing. Such devices include, for example, modem cards, wireless cards, network interface cards, sound cards, or other types of controllers for any of a variety of known input devices. The output controller can include a controller for any of a variety of known display devices for presenting information to a user, whether human or machine, local or remote. In the presently described embodiment, the functional elements of the computer communicate with each other through a system bus. Some computer embodiments can communicate with some functional elements using a network or other type of remote communication. As will be apparent to those skilled in the relevant art, device control and / or data processing applications, when implemented in software, can be loaded and executed from the system memory and / or a memory storage device. All or part of the device control and / or data processing applications may be resident in a similar device such as a read-only memory or a memory storage device, and such a device does not require that the device control and / or data processing applications be first loaded via an input / output controller. It will be understood by those skilled in the relevant art that, for the sake of advantageous execution, the device control and / or data processing applications, or parts thereof, may be loaded by a processor into the system memory, or the cache memory, or both, in a known manner. Also, the computer can include one or more library files, experimental data files, and Internet clients, and may be stored in the system memory. For example, the experimental data can include data related to one or more experiments or assays, such as detected signal values, or other values associated with one or more sequencing by a synthesis (SBS) experiment or process.In addition, the Internet client may include applications that can be used to access remote services on other computers using the network, and may further include, for example, what is commonly referred to as a "web browser". In this example, some commonly adopted web browsers include Microsoft Internet Explorer from Microsoft Corporation, Mozilla Firefox from Mozilla Corporation, Safari from Apple Computer Corp, Google Chrome from Google Corporation, or other types of web browsers currently known or to be developed in the art. Microsoft Internet Explorer that can be obtained, Mozilla C orporation's Mozilla Firefox, Apple Computer Corp's Safari, Google Corporation's Google Chrome, or other types of web browsers currently known or to be developed in the art. In the same or other embodiments, the Internet client may include special software applications that can be used to access remote information through the network, such as data processing applications for biological applications, or can be elements thereof.

[0062]

[0087] The network can include one or more of many different types of networks well known to those skilled in the art. For example, the network may include a local or wide area network that can employ what is generally referred to as the TCP / IP protocol suite for communication. The network may include a network that constitutes a worldwide system of interconnected computer networks, generally referred to as the Internet, or can also include various intranet architectures. Also, among users in a network connection environment, some prefer to employ what is usually called a "firewall" (sometimes also called a packet filter or border protection device) to control information traffic to and from hardware and / or software systems. For example, a firewall may include a hardware or software element or some combination thereof and is typically designed to enforce security policies introduced by a user, such as, for example, to a network administrator, etc.

[0063]

[0088] In one embodiment of the system 100 of FIG. 1, a sample is attached to a substrate holder (not shown in FIG. 1. Examples of sample substrate holders that can be part of the system 100 include a heater bas, a Peltier heating and cooling bath, and a tray for holding multiple samples. In one embodiment, the entire tray of samples is loaded into the system 100 by the user) and a substrate transfer / substrate movement system is used to fix the sample to a microscope slide. It is directed towards the baking / drying oven 112 by the conveyer 106. In other embodiments, the baking / drying of the sample is performed outside the system 100 and is directly loaded onto the sample substrate holder by the user of the system 100. Alternatively, although will be described in more detail with respect to subsequent figures, the sample can be placed on the sample substrate holder, and all systems required to execute a specific staining protocol can be moved towards the sample using, for example, an actuator associated with the bulk fluid applicator system 116 or the microfluidic applicator system 118. The reagents used in the fluid applicator system 116 or the microfluidic applicator system 118 can, in certain embodiments, be moved from the reagent storage unit 120 using the reagent transfer system 122 and can be fluidly connected to the fluid applicator system 116 or the microfluidic applicator system 118.

[0064]

[0089] In certain staining protocols, it may be desirable to perform antigen activation or target activation on the sample before another processing step can be carried out. In one embodiment, the substrate transfer / substrate movement system 106 is used to direct the sample towards the antigen / target activation system 124. The antigen / target activation system 124 can be, for example, an enclosed chamber that can be pressurized and heated to a temperature higher than the normal boiling point of water. Optionally, the bulk antigen activation solution can be supplied to the antigen / target activation system 124 by the fluid / air supply module 126. In certain embodiments, the antigen / target activation system can be configured to be optimized for antigen activation (unmasking of antigenic tissue prior to IHC) or target activation (unmasking of nucleic acid sequences prior to ISH ). In other embodiments, the disclosed antigen / target activation system 124 can include one or more dedicated antigen activation subsystems and one or more dedicated target activation subsystems.

[0065]

[0090] Alternatively, an antigen-activating solution and an overlying layer of a low volatility organic solvent can be dispensed onto the sample by the bulk fluid applicator system 116, with or without performing stirring / mixing using agitation such as gas injection or vibration and heating the sample by a heater base that is part of the sample substrate holder. As an alternative to the overlying layer of the low volatility organic solvent, an opposable surface can be placed over the antigen-activating solution, which can serve to reduce evaporation and, in certain embodiments, can be moved to mix the fluid covering the sample.

[0066]

[0091] Following treatment with the bulk fluid, in certain embodiments, it may be desirable to use the air knife system 128 to remove the bulk fluid from the sample and direct this fluid to the waste management system 130. The waste management system 130 can include one or more reservoirs for capturing waste fluids generated by different protocol steps of a given staining protocol. The waste management system 130 can also include mechanisms for treating, separating, and / or filtering the waste fluids.

[0067]

[0092] As shown in FIG. 1, a microfluidic applicator management system 132 is provided to ensure that the microfluidic applicator 118 remains functional. For example, the microfluidic applicator management system 132 can include a washing station or can simply be a location where the microfluidic applicator system 118 can be moved when not in use to ensure that it dries out and does not become clogged during periods when the microfluidic reagent applicator (not shown in FIG. 1) of the microfluidic applicator system 118 is not being used.

[0068] ​​​

[0093] Further, once their processing protocols are complete, the cover slip module 134 can also be made part of the system 100 for placing the cover slip over the sample.

[0069]

[0094] FIG. 2 shows an embodiment of the disclosed system 200, where the sample support substrate 202 is continuously moved through the processing modules 206, 208, 210, 212, 214, and 216 on the conveyor 204. The modules 206, 208, 210, 212, 214, and 216 are used to process the sample, i.e., these modules may be used to perform one or more process steps required for a particular staining protocol. In certain embodiments, at least one of the processing modules 206, 208, 210, 212, 214, and 216 includes a microfluidic reagent applicator, and at least one of the processing modules 206, 208, 210, 212, 214, and 216 includes a bulk fluid applicator. In one embodiment, at least one of the processing modules 206, 208, 210, 212, 214, and 216 is stationary and held so as to impart relative movement to the sample passing through this at least one processing module (continuously at a constant speed, variably, or in a stop-start manner). In other embodiments, the processing modules 206, 208, 210, 212, 214, and At least one of the calls 216 is connected to an actuator (not shown) that can impart relative motion in any combination of the x, y, and z coordinate directions. For example, in certain embodiments, the conveyor 204 operates in a stop-start manner under the control of the control system 102 to continuously transport the sample support substrate 202 in the vicinity of the processing modules 206, 208, 210, 212, 214, and 216. Once the sample support substrate is positioned in the vicinity, the actuator moves the processing module relative to the substrate to dispense one or more fluids onto the sample. In other specific embodiments, at least one of the processing modules 206, 208, 210, 212, 214, and 216 includes a fluid aspirator. In other specific embodiments, at least one of the processing modules includes an air knife. In still other specific embodiments, at least one of the processing modules includes two or more of a bulk fluid applicator, a microfluidic reagent applicator, a fluid aspirator, and an air knife. When not in use, all of the processing modules 206, 208, 210, 212, 214, and 216 can be moved to the reagent storage unit 220.

[0070]

[0095] Figure 3 shows a schematic diagram of an embodiment of the disclosed system 300, including an array of stationary sample substrate holders 302 and a plurality of processing modules 304 / 306, 308, and 310. Also shown are a reagent storage module 312 and a microfluidic applicator management system 34. In this embodiment, the processing module 304 / 306 includes a bulk fluid dispensing system, and the bulk fluid applicator module 306 is fluidly connected to the fluid / air / vacuum supply module 304 via one or more flexible fluid connections 316, 318, and 320. In certain embodiments, the flexible fluid connections 316, 318, and 320 supply bulk fluid, air, and vacuum, respectively, to the bulk fluid applicator module 306. In more specific embodiments, the fluid / air / vacuum supply module 304 includes a plurality of bulk fluid reservoirs that can be switched to supply different fluids to the bulk fluid applicator module 306. In yet more specific embodiments, the flexible fluid connections 316 and 320 supply compressed air to an air knife mounted on the bulk fluid applicator module 306 and supply vacuum to a fluid aspirator mounted on the bulk fluid applicator module 306.

[0071]

[0096] In one embodiment, and referring again to FIG. 3, processing module 308 comprises a microfluidic reagent applicator dedicated to supplying a detection chemical reaction reagent to a sample placed on a sample substrate holder inside array 302, and processing module 310 supplies a reagent, for example, a primary antibody. In this embodiment, processing module 310 moves to reagent storage module 312 (which can be cooled and / or humidified) and retrieves a microfluidic reagent applicator that includes an integrated reagent reservoir corresponding to the primary antibody that is to be dispensed to a particular sample in the array according to the planned staining protocol. Once the dispensing of the primary antibody is complete, this applicator is returned to reagent storage module 312, perhaps after being sent to microfluidic applicator management system 314 for cleaning / absorbing or humidifying (such as by application of a moisturizing agent). Processing module 308 in this embodiment can include one microfluidic reagent applicator fluidly connected to a plurality of integrated reagent reservoirs, as shown in FIG. 8.

[0072]

[0097] In other embodiments, and referring again to FIG. 3, sample substrate holder array 302 can be an array such as a 1×n, 2×n, 3×n, n×n array, for example, a 1×20 array, a 2×15 array, or a 3×10 array for supplying a total of 30 sample substrate holders. The system can also have other numbers of sample substrate holders, such as from 5 to 200 substrate holders, and these can be arranged in any possible array configuration to reach the total number of sample substrate holders. Some or all of these substrate holders can be provided with a heater - based or Peltier heating / cooling - based unit.

[0073]

[0098] 4 illustrates an embodiment of a bulk fluid applicator module 400. Bulk fluid applicator module 400 includes a body 402 (which may include a reservoir, and intermediate reservoir, or may simply provide a fluid connection to a bulk fluid / vacuum supply module (not shown)), a bulk fluid applicator needle 404, and a fluid aspirator needle 406, which when used together may form a fluid knife 408 between the needles. According to the disclosed methods, the entire bulk fluid applicator module may be moved across a sample support substrate 410 to treat all or a portion of a sample.

[0074]

[0099] 4B illustrates a second embodiment of a bulk fluid applicator module 420. The bulk fluid applicator module 420 includes a body 422, a vacuum nozzle 424, a bulk fluid applicator nozzle 426, and an air knife nozzle 428. An actuator 430 can be used to move the entire module 420 over all or a portion of the sample support substrate 410.

[0075]

[0100] FIG. 5A illustrates a bulk fluidic array configured for treatment of a sample disposed on a substrate. 5A shows another embodiment of an applicator module 500. In this embodiment, the module includes a body 510, a bulk dispensing needle 502, a fluid aspirating needle 504, a bulk fluid inlet 508, and a vacuum port 506 for aspirating waste fluids away from the sample (such as into the waste management system 130 of FIG. 1). The embodiment of FIG. 5A is configured to treat an entire slide or a portion of a sample (depending on the separation between the needles), such as to form square or rectangular wells in the paraffin of a paraffin-embedded tissue sample.

[0076]

[0101] FIG. 5B illustrates yet another embodiment of a bulk fluid applicator module 520. is shown. The bulk fluid applicator module 520 includes a body 522, a bulk dispensing needle 524, a fluid aspiration needle 526, a bulk fluid inlet 528, and a vacuum port 530 for aspirating waste fluid from the sample away (such as to the waste management system 130 of FIG. 1). The embodiment of FIG. 5B can be rotated in any one or any combination of directions in the x, y, and z coordinate directions in addition to being moved by an actuator as shown. Also, the bulk fluid applicator module of this embodiment can treat the entire slide or a part of the sample (depending on the separation between the needles), for example, to form square wells or rectangular wells in the paraffin of paraffin-embedded tissue samples. However, since the entire module can also be rotated, it is possible to treat a part of the sample in a circular pattern, for example. For example, the bulk fluid applicator module 520 can be used to form circular wells or wells of a similar shape in the paraffin of paraffin-embedded tissue samples.

[0077] is configured to treat the entire slide or a part of the sample (depending on the separation between the needles), for example, to form square wells or rectangular wells in the paraffin of paraffin-embedded tissue samples. However, since the entire module can also be rotated, it is possible to treat a part of the sample in a circular pattern, for example. For example, the bulk fluid applicator module 520 can be used to form circular wells or wells of a similar shape in the paraffin of paraffin-embedded tissue samples.

[0077]

[0102] FIG. 6A shows an embodiment of a microfluidic reagent applicator 600 according to the present disclosure. In this embodiment, reagent reservoirs 602a, 602b, 602c, and 602d are integrated with corresponding droplet-on-demand microfluidic actuators 604a, 604b, 604c, and 604d. The microfluidic reagent applicator 600 can be disposable or refillable and is shown as an assembly of four individual applicators, but one or more (many more) (such as corresponding to each step of a staining protocol. This Numbers of 5 or more, 10 or more, or 20 or more) can be combined into one unit and used, for example, with the substrate transfer / substrate movement system 204 of FIG. 2. Alternatively, an aggregate of individual applicators can be used to treat multiple samples simultaneously, for example, even if there are 5 or more, 10 or more, or 20 or more, and a part or all of the aggregate of substrate-mounted samples held in a tray for holding such substrate-mounted samples can be treated simultaneously. The microfluidic reagent applicator according to this embodiment can be added to and removed from an automated staining system in an overall manual process, or can be used in combination with a reagent transfer system for loading and unloading through an access port in an enclosure covering the staining system.

[0078]

[0103] FIG. 6B shows another embodiment of the microfluidic reagent applicator 610 according to the present disclosure. In this embodiment, replaceable reagent reservoirs 612a, 612b, 612c, and 612d are fluidly coupled to corresponding droplet-on-demand microfluidic actuators 614a, 614b, 614c, and 614d. The microfluidic reagent applicator reservoirs 612a, 612b, 612c, and 612d can be disposable or refillable. and shown in FIG. 6B as a collection of four individual applicators, but one or more (such as corresponding to each step of the staining protocol; this number can also be 5 or more, 10 or more, or 20 or more) can be combined into one unit and used, for example, with the substrate transfer / substrate movement system 204 as shown in FIG. 2. Alternatively, the collection of individual applicators can be used to treat multiple samples simultaneously, for example, 5 or more, 10 or more, or 20 or more, and can be used to treat some or all of the collection of substrate-mounted samples held in a tray for holding such substrate-mounted samples. The replaceable reagent reservoirs 612a, 612b, 612c, and 612d can be added to and removed from the automated staining system in an overall manual process, or can be used in combination with a reagent transfer system for loading and unloading through access ports in an enclosure covering the staining system.

[0079]

[0104] FIG. 6C shows yet another embodiment of the microfluidic reagent applicator 620 according to the present disclosure. In this embodiment, remote reagent reservoirs 622a, 622b, 622c, and 622d are connected through fluid pipelines 626a, 626b, 626c, and 626d to the corresponding It is fluidly coupled to droplet-on-demand microfluidic actuators 624a, 624b, 624c, and 624d. The microfluidic reagent applicator / reservoirs 622a, 622b, 622c, and 622d can be disposable or refillable, and are shown in FIG. 6C as an assembly of four individual applicators, but one or more (such as corresponding to each step of the staining protocol. This number can also be 5 or more, 10 or more, or 20 or more) can be combined into one unit and used, for example, with the substrate transfer / substrate movement system 204 of FIG. 2. Alternatively, an assembly of individual applicators can be used to treat multiple samples simultaneously, for example, even if there are 5 or more, 10 or more, or 20 or more, and can be used to simultaneously treat some or all of an assembly of substrate-mounted samples held in a tray for holding such substrate-mounted samples. The fluid pipelines 626a, 626b, 626c, and 626d can also be rigid, flexible, or a combination of both rigid and flexible, both between pipelines and within a single pipeline. Also, the embodiment of FIG. 6C is also suitable for simultaneously processing all or part of an array of samples, as shown in FIG. 3, when the pipelines are flexible. Also, such a configuration also provides an opportunity to refill the reservoir from a location external to the device, and thus can be said to be particularly suitable for dispensing bulk fluids.

[0080]

[0105] FIG. 6D shows yet another embodiment of the microfluidic reagent applicator 630 according to the present disclosure. In this embodiment, remote reagent reservoirs 632a, 632b, 632c, and 632d are fluidly coupled to corresponding droplet-on-demand microfluidic actuators 634a, 634b, 634c, and 634d through fluid pipelines 636a, 636b, 636c, and 636d. Also, this embodiment includes intermediate reservoirs 638a, 638b, 638c, and 638d. The microfluidic reagent applicator / reservoirs 632a, 632b, 632c, and 632d can be disposable or refillable, In FIG. 6D, it is shown as an assembly of four individual applicators, but one or more (such as corresponding to each step of the staining protocol. This number can also be 5 or more, 10 or more, or 20 or more) can be combined into one unit and used, for example, with the substrate transfer / substrate movement system 204 of FIG. 2. Alternatively, the assembly of individual applicators can be used to treat multiple samples simultaneously. For example, even if there are 5 or more, 10 or more, or 20 or more, it can be used to simultaneously treat some or all of the substrate-mounted samples held in a tray for holding such substrate-mounted samples. The fluid pipelines 636a, 636b, 636c, and 636d can also be rigid, flexible, or a combination of both rigid and flexible, both between the pipelines and within one pipeline. Also, the embodiment of FIG. 6C is also suitable for simultaneously processing all or part of an array of samples when the pipeline is flexible, as shown in FIG. 3. Also, such a configuration also provides an opportunity to refill the reservoir from a location outside the device. Therefore, it can be said that it is particularly suitable for dispensing bulk fluids. Furthermore, due to the intermediate reservoirs 638a, 638b, 638c, and 638d, the remote reagent reservoirs 632a, 632b, 632c, and 632d can be easily replaced during operation without interrupting the processing of samples in the automated staining system.

[0081]

[0106] Any combination of the microfluidic reagent applicators shown and described with respect to FIGS. 6A - 6D can also be employed in one automated biological sample staining system according to the present disclosure.

[0082]

[0107] FIG. 7 shows a replaceable fluid reservoir 704 (shown in FIG. 6B and described with reference to FIG. 6B) FIG. 7 schematically shows a reagent transfer system 700 that can be used to transfer reagents in and out of a reagent storage unit 720 when once disconnected from a microfluidic actuator 702 (as described with reference to FIG. 6A). The reagent storage unit 720 can be cooled to extend the life of the reagent while not in use. Also shown is how a microfluidic reagent applicator (as shown in FIG. 6A and described with reference to FIG. 6A) including an integrated reservoir 706 can be reciprocated in and out of the reagent storage unit 720 by the reagent transfer system 700. Further, FIG. 7 illustrates how a remote reagent reservoir 708 can be held in a storage state and used to supply a microfluidic reagent applicator 710 through a pipeline 712 and an optional intermediate reservoir (as shown in FIGS. 6C and 6D and described with reference to FIGS. 6C and 6D) as needed. FIG. 8 shows a microfluidic reagent applicator system 800 including a droplet-on-demand actuator head 802 fluidly connected to a plurality of reagent reservoirs 804a, 804b, 804c, and 804d. Also shown in FIG. 8 is an embodiment of a sample substrate holder 806, in which a substrate 808 supporting a sample 810 is placed on a base 812. This base can be a heater base or a Peltier heating / cooling base. If compatible, the reagents contained in the plurality of reagent reservoirs 804a, 804b, 804c, and 804d can be sequentially dispensed to perform a staining protocol on the sample 810. Such a simple configuration is suitable for a point-of-care system, such as a system for staining a frozen tissue sample in an operating room. Such a point-of-care system can be, for example, used by a surgeon to take a tumor from a patient beyond its resection margins.

[0083]

[0108] FIG. 8 shows a microfluidic reagent applicator system 800 including a droplet-on-demand actuator head 802 fluidly connected to a plurality of reagent reservoirs 804a, 804b, 804c, and 804d. Also shown in FIG. 8 is an embodiment of a sample substrate holder 806, in which a substrate 808 supporting a sample 810 is placed on a base 812. This base can be a heater base or a Peltier heating / cooling base. If compatible, the reagents contained in the plurality of reagent reservoirs 804a, 804b, 804c, and 804d can be sequentially dispensed to perform a staining protocol on the sample 810. Such a simple configuration is suitable for a point-of-care system, such as a system for staining a frozen tissue sample in an operating room. Such a point-of-care system can be, for example, used by a surgeon to take a tumor from a patient beyond its resection margins. For performing a staining protocol on the sample 810, the reagents contained in the plurality of reagent reservoirs 804a, 804b, 804c, and 804d can be sequentially dispensed. Such a simple configuration is suitable for a point-of-care system, such as a system for staining a frozen tissue sample in an operating room. Such a point-of-care system can be, for example, used by a surgeon to take a tumor from a patient beyond its resection margins. It can be used to determine whether removal has been successful. The reagent reservoirs 804a, 804b, 804c, and 804d can be integrated with the droplet-on-demand actuator head 802, can be made interchangeable with the droplet-on-demand actuator head 802, or can be located remotely and fluidly connected through pipelines.

[0084]

[0109] FIG. 9A shows a front view of an embodiment of an integrated system, which integrated system includes a microfluidic droplet-on-demand actuator, a bulk fluid applicator slit, and a fluid aspirator slit. As also shown in FIG. 9B, a reagent reservoir 902 is fluidly coupled to the microfluidic reagent applicator head 906. As shown in the front view of FIG. 9A and the side view of FIG. 9B, a dual slit combination bulk fluid applicator and fluid aspirator 904 are mechanically coupled to the microfluidic reagent applicator head 906. As shown in the front view of FIG. 9A, the integrated system includes a bulk fluid inlet 908 and a fluid suction port 910, and the fluid suction port 910 is also shown in the side view of FIG. 9B. FIG. 9C is a bottom view of this integrated system, showing the dual slit combination bulk fluid applicator and fluid aspirator 904 including a bulk fluid applicator slit 912 and a fluid aspirator slit 914. Also shown in FIG. 9C is the microfluidic droplet-on-demand array 916 of the microfluidic reagent applicator head 906.

[0085]

[0110] FIG. 10A shows a front view of an embodiment of an integrated system, which embodiment is micro It includes a microfluidic droplet on-demand actuator, a bulk fluid applicator needle, and a fluid aspirator needle. As also shown in FIG. 10B, a reagent reservoir 1002 is fluidly coupled to a microfluidic reagent applicator head 1006. As shown in the front view of FIG. 10A and the side view of FIG. 10B, a dual needle composite bulk fluid applicator and fluid aspirator 1004 are mechanically coupled to the microfluidic reagent applicator head 1006. As shown in the front view of FIG. 10A, this integrated system includes a bulk fluid inlet 1008 and a fluid suction port 1010, and the fluid suction port 1010 is also shown in the side view of FIG. 10B. As shown in the front view of FIG. 10A, this integrated system includes a bulk fluid applicator needle 1012 and a fluid suction needle 1014, and the fluid suction needle 1010 is also shown in the side view of FIG. 10B. FIG. 10C shows a bottom view of this integrated system, a dual needle composite bulk fluid applicator and fluid aspirator including the bulk fluid applicator needle 1012 and the fluid aspirator needle 1014 1004 is shown. Also shown in FIG. 10C is a microfluidic droplet on-demand array 1016 of the microfluidic reagent applicator head 906.

[0086]

[0111] FIG. 11 is for use alone or additional such same ones and this Shown is one dyeing subsystem 1000 for use in combination with other subsystems according to the disclosure. In this embodiment, the microfluidic reagent applicator 1102 is mechanically coupled to the actuator 1104. In certain embodiments, a substrate holder 1108 that holds the sample support substrate 1110 is also included. The substrate holder 1108 is also mechanically coupled to the actuator 1112. In certain embodiments, a composite bulk fluid applicator and air knife module 1106 is also included. One or more waste collection units 1114 (which can be fluidly connected to a waste management system) are also shown. In the loading position, the substrate holder 1108a holds the sample support substrate 1110a in a horizontal position, and then the actuator 1104 guides (bring) the microfluidic reagent applicator 1102 to a fixed position in order to dispense fluid onto the sample. Depending on which reagent is dispensed onto the sample, the actuator 1112 moves the substrate holder and the substrate to one of at least two different positions 1108b, 1110b, or 1110c so that the composite bulk fluid applicator and air knife (such as at positions 1106a, 1106b, or 1106c or any intermediate position not shown) can, for example, pour a rinse solution and then move or "blow away" this rinse solution and any reagent residue and remove them from the sample to their respective waste collection units 1114a, 1114b. Note that in certain embodiments, the bulk fluid applicator and air knife move with the substrate holder, while in other embodiments, the bulk fluid applicator and air knife are separate units and are not connected to the substrate holder (and thus can move in and out), which will be recognized by those skilled in the art. In an alternative embodiment, the entire row of microfluidic reagent applicators can be translated backward along the axis of rotation of the actuator 1104 so that additional reagents can be guided to the dispensing / loading position relative to the sample. In FIG. 11, the dispensing position is shown horizontally, but alternative configurations are possible where the sample support substrate is held in a non-horizontal position, such as between approximately 1 degree and approximately 90 degrees from horizontal, or held upside down during the dispensing of fluid from the microfluidic reagent applicator.

[0087]

[0112] FIG. 12 shows a top view of the embodiment of FIG. 11, where the composite bulk fluid applicator / air knife module is shown as 1206. The substrate holder 1208 holds the sample support substrate 1210, and the module 1206 is moved over the entire area of the sample support substrate 1210 by a screw drive 1224 powered by a motor 1228. Also, the module 1206 is guided by rails 1226 and held in place by struts 1230. Air is supplied to the module 1206 through port 1220, and bulk fluid is supplied to the module 1206 through port 1222.

[0088]

[0113] FIG. 13 shows a top view of the embodiment of FIG. 11, but in this embodiment 1300, there are additional functions of the bulk fluid applicator / fluid aspirator / air knife module 1306. In this embodiment, at least two different reagents 1346 and 1348 are supplied to the microfluidic reagent applicator 1302. The composite bulk fluid applicator / fluid aspirator / air knife module is supplied with a vacuum 1340, compressed air 1342, and at least one bulk reagent 1344. The actuator 1312 enables the use of the arrangement of FIG. 13 in the embodiment of FIG. 11.

[0089]

[0114] FIG. 14 shows a particular embodiment of a substrate holder system 1400 that can be used in combination with a microfluidic reagent dispenser to form a chamber above a sample held on a substrate. Here, the microfluidic reagent actuator 1404 ​​​​It fits with the substrate holder 1406 and further fits with the sealants 1412a and 1412b. Shown inside are the substrate holder 1408 and the sample support substrate 1410. In certain embodiments, the microfluidic reagent actuator is a thermal droplet-on-demand system. In a particular embodiment, the fluid reservoir 1402 contains the antigen activation solution, and the antigen activation solution is dispensed onto the sample within the chamber. In certain embodiments, and assuming vents are included, the contents dispensed onto the sample may be heated and then replenished with additional fluid. In other embodiments, the chamber can be pre-pressurized or the pressure can increase within the chamber as heat is applied.

[0090]

[0115] The applicant has discovered the surprising fact that, using the systems and methods of the present disclosure, hematoxylin and eosin, which are generally incompatible in solution, can be co-dispensed. Compared to linear assays, co-dispensing using the systems and methods disclosed herein enables a significant reduction in the number of assay steps (e.g., from 10 steps to 5 steps) and a reduction in the total amount of assay attendant (e.g., from about 2.44 mL / slide to about 1.26 mL / slide), while obtaining similar results. fact. Compared to linear assays, co-dispensing using the systems and methods disclosed herein enables a significant reduction in the number of assay steps (e.g., from 10 steps to 5 steps) and a reduction in the total amount of assay attendant (e.g., from about 2.44 mL / slide to about 1.26 mL / slide), while obtaining similar results.

[0091]

[0116] FIGS. 15A and 15B show two embodiments of deparaffinization according to the present disclosure which were comparable to standard methods with respect to incubation time and volume. The figures show printed deparaffinization. That is, it shows the printing of organic and transfer fluids onto paraffin sections, followed by bulk washing with a solution. It is possible to have co-printing or sequential modes It was found that the assay required only small volumes (~100uL / in^2) and little or no incubation time (~2 minutes to print). A single microfluidic deparaffinization process can also be utilized using dual needles and dual slits (as described herein), again requiring only small volumes and little or no incubation time (~2 minutes to print).

[0092]

[0117] Above, bulk fluid applicators, fluid applicators, and air knives Although specific configurations have been disclosed, other configurations are possible. For example, the system described in US8883509 can be used in combination with other features disclosed herein. Similarly, the specific bulk fluid handling and reagent dispensing mechanisms disclosed in WO2015 / 086534 can be used in combination with other features disclosed herein.

[0093]

[0118] Additional Embodiments

[0094]

[0119] The various components shown in FIG. 16 are described below. In one embodiment, the control The control system or method includes a "print command and map generator" that allows for the directed delivery of droplets to obtain the desired image on the droplet target. It functions to place the nozzles in a coordinated fashion to produce a combinatorial 2- (or 3-) axis motion that coordinates with the firing of the nozzles. Typically, this is a mapping between a binary (or monochrome) bitmap file, where one state (i.e. logical true) of each pixel in the file corresponds to one drop being dispensed from one nozzle at a particular location. Conversely, a logical false state corresponds to not firing at that location during the print routine. The output of this function is the commands to the motion system, which in turn are commands to the print system.

[0095]

[0120] In one embodiment, the control system or control method includes a "Digital Firing Pulse Generator". Its function is to decompose the print map in the context of both the relative motion system and the assignment of individual droplet dispensing operations to the corresponding nozzles. In one embodiment, the control system or control method includes a "relative motion system". This facilitates the target motion of the print head by a set of commands (i.e., print commands) sent from the print instruction and the map generator. To monitor the progress in the mapping of the image to the physical print and correspondingly to monitor when to send out the droplets to be deposited on the print droplet target, information about the motion of the system relative to the reference position is relayed to the digital firing pulse generator via a step counter.

[0096]

[0121] In one embodiment, the control system or control method includes a "relative motion system". This facilitates the target motion of the print head by a set of commands (i.e., print commands) sent from the print instruction and the map generator. To monitor the progress in the mapping of the image to the physical print and correspondingly to monitor when to send out the droplets to be deposited on the print droplet target, information about the motion of the system relative to the reference position is relayed to the digital firing pulse generator via a step counter.

[0097]

[0122] In one embodiment, the control system or control method includes a "step counter". The step counter provides the function of making the digital firing pulse generator understand the motion and relative positions of the target and the print head in order to train the deposition of droplets by the relative motion system.

[0098]

[0123] In one embodiment, the control system or control method includes a "waveform generator". When a logical true is issued for a specific nozzle address, the act of droplet dispensing is initiated. This function converts the signal into the analog signals required to prepare, dispense, and refill the nozzle for successful ejection of the droplet.

[0099]

[0124] In one embodiment, the control system or control method includes an "amplifier". The raw The generated waveform is amplified to an appropriate range of excitation potential (i.e., voltage or energy) to induce ejection of the droplets.

[0100]

[0125] In certain embodiments, the control system or control method includes a "fluid droplet dispenser". The amplified signal is passed to the corresponding nozzle, from which droplets are fired onto the droplet target in synchrony with the relative motion system.

[0101]

[0126] In certain embodiments, the control system or control method includes a "droplet target". The ultimate target that receives droplets from the fluid droplet dispenser is coordinated and moved by the relative motion system to map a print map onto this target.

[0102]

[0127] Describe the variety of physical control methods outlined in FIG. 17. In certain embodiments, the control system or control method includes a CPU. In this case, the CPU is a desktop computer. The role of the CPU is to coordinate the issuance of commands to the physical system through print instructions and a print map. These are obtained from a monochrome bitmap file, and the desired droplet print density is obtained for the x and y axis directions. The x-axis print density is determined physically by setting the encoder step size for the Print Manager Board, and the y-axis print density is determined by adjusting the saber angle of the print head. Manager Board), and the y-axis print density is determined by adjusting the saber angle of the print head. by adjusting the saber angle of the print head. It is set like this. For example, an image of 1000×1000 pixels printed at 1000×1000 dpi generates a 1 in2 print. Similarly, when the same image is printed at 500×500 dpi, a 2 in2 print is generated. Finally, when the same image is printed at 2000×2000 dpi, a 0.5 in2 print is generated. As part of the print command, the CPU provides speed information to the relative motion system and further assigns a y-step and a start position for the print job. By fixing the printing system to a "raster printing" configuration, the image is converted into a print on the print target by a continuous print movement in the x direction prior to the next print pass in the x direction and a subsequent incremental y-step movement.

[0103]

[0128] In one embodiment, the control system or control method includes information about the print command, relative motion system and the print job for adjusting the print manager board.

[0104]

[0129] In one embodiment, the control system or control method includes a monochromatic (i.e., binary) bitmap file where logical true corresponds to droplet dispensing and logical false corresponds to lack of droplet dispensing. The print map is scaled using the x and y print densities when being converted from the pixel map to the actual print (as previously explained in the CPU section).

[0105]

[0130] In one embodiment, the control system or control method is the print manager · It includes a board and performs the role of the digital emission pulse generator function in the previous chapter. The board decomposes the print map and assigns nozzles for emission to specific pixels in the map. When information about the encoder step size is given, it determines how many encoder pulses should be awaited between each pixel in the print map. Generally, there is a one-to-many relationship between this board and the drive card, and the drive card acts as a daughter board for the higher-level functions actually executed on the print manager board.

[0106]

[0131] In certain embodiments, the control system or control method includes a relative motion system as described above in the previous chapter.

[0107]

[0132] In certain embodiments, the control system or control method includes a digital emission signal. When the print manager board confirms that the droplet dispensing operation must be performed (by decomposing the print map and monitoring the print head position using the encoder step pulse as feedback), it issues the emission signal to the nozzle drive card downstream of the corresponding data path, thereby issuing a command to the nozzle (or multiple nozzles simultaneously).

[0108]

[0133] In certain embodiments, the control system or control method includes a drive card and performs the functions of the waveform generator and amplifier described in the previous chapter. Its roles include power management, eliminating crosstalk between the electrical paths of the firing nozzles, and returning the print nozzles to the ready state for re-emission. Generally, it is mapped one-to-one with the print head, and the drive card provides a dedicated signal line for each nozzle on the print head. However, the actual architecture can also support a one-to-many association with the print head. Because each signal line is mapped to the nozzle ​ This is because they are flip-flopped and it does not matter whether these nozzles are co-located on the same print head.

[0109]

[0134] In one embodiment, the control system or control method includes vector graphics. In other embodiments, a plotter approach and a vector graphics (e.g., PostScript) language for describing the printing operation can provide certain advantages. Vector graphics describe lines and arcs for printing instead of pixels. This enables infinite magnification and transformation of the printed image without sacrificing resolution. Also, this enables complex coordinate-based movement of the printing system. The printing operation can be determined by programming (functions, loops, variables, etc.) rather than being statically determined by an image file. In this environment, for example, print vectors can be defined for specific lengths, speeds, firing frequencies, and widths of the print swath. It is possible.

[0110]

[0135] The advantages (as shown in FIG. 17) refer to a common framework for describing all motion components for any operation (washing, drying, etc.) that the stainer has to perform. It can handle complex two- or three-axis printing operations. There is no need to sequentially perform printing operations in the x direction followed by steps in the y direction. It is easy to expand and transform the image of the tissue to the printing area, and it is easy to reuse the image from one slide to print other stains on other slides by cutting the same tissue.

[0111]

[0111]

[0136] Multi-color bitmap. For each print head and each printed image Instead of individual monochrome bitmaps being applied to the samples, this information is encoded into a more complex image data file. For example, a print run may be encoded as a 64-bit bitmap, allowing eight time-sequenced print patterns to be mapped to eight unique print heads. This is a very efficient way to encode an entire complex assay in a very dense format. This can accommodate a large number of printed reagents, simultaneous print runs, ratiometric printing operations, and gradient printing operations.

[0112]

[0137] The advantage (as shown in Figure 17) is the simultaneous use of multiple print heads. This refers to a precise method for defining a time series or describing the time redispensing of reagents by print. By using common image storage languages ​​such as JPEG, TIFF, JPEG-2000, etc., it can be compressed as opposed to bitmap file formats.

[0113]

[0138] Various other modifications of the disclosed systems and methods are described herein. In addition to those described above, modifications will be apparent to those skilled in the art, and such modifications are intended to fall within the scope of the appended claims. For example, while the above disclosure has focused on the treatment of cell and tissue samples mounted on microscope slides, the disclosed systems and methods, in their various embodiments, are equally applicable to the preparation of other types of biological samples on other types of substrates, such as the preparation of microarrays of nucleic acids or antibodies or target biological samples for mass spectrometry analysis, or the preparation of hematological samples. Each of the references cited in this application is incorporated herein by reference in its entirety to the extent that it is not inconsistent with this disclosure.

Claims

1. An automated biological sample staining system, at least one microfluidic reagent applicator for discharging a staining reagent, the at least one microfluidic reagent applicator being fluidly connected to at least one first reagent reservoir integrated within the microfluidic reagent applicator, at least one microfluidic reagent applicator; at least one bulk fluid applicator for dispensing a deparaffinizing reagent to a sample placed on a substrate, the at least one bulk fluid applicator being fluidly connected to one or more refillable second reagent reservoirs, at least one bulk fluid applicator; at least one fluid aspirator attached to a module of the at least one bulk fluid applicator, the at least one fluid aspirator being combined with the at least one bulk fluid applicator into at least one first type of reagent management unit, at least one fluid aspirator; at least one sample substrate holder; at least one relative motion system configured to be coupled to the at least one first type of reagent management unit, the at least one relative motion system enabling relative movement between the first type of reagent management unit and the at least one relative motion system, at least one relative motion system; a control system programmed to execute at least one staining protocol on the sample placed on the substrate and held by the at least one sample substrate holder, the control system controlling the at least one first type of reagent management unit, the at least one sample substrate holder, and the at least one relative motion system to execute individual steps of the at least one staining protocol, control system; A system comprising the above components.

2. The system according to claim 1, further comprising at least one sample imaging system.

3. The system according to claim 1, further comprising at least one air knife.

4. In the system according to claim 1, the at least one microfluidic reagent applicator, the at least one bulk fluid applicator, and the at least one fluid aspirator are combined into at least one second type of reagent management unit. A system, wherein the second type of reagent management unit is coupled to the at least one relative motion system and configured to enable relative motion between the second type of reagent management unit and the at least one relative motion system.

5. In the system according to claim 3, the at least one microfluidic reagent applicator, the at least one bulk fluid applicator, and the at least one air knife are combined with at least one third type of reagent management unit, A system, wherein the third type of reagent management unit is coupled to the at least one relative motion system and configured to enable relative motion between the third type of reagent management unit and the at least one relative motion system.

6. A system according to claim 1, further comprising at least one waste management system.

7. A system according to claim 1, further comprising at least one sample identification system communicatively coupled to the control system.

8. In the system according to claim 1, the at least one bulk fluid applicator and the at least one fluid aspirator comprise a pair of needles.

9. In the system according to claim 8, the pair of needles are separated by at least 0.1 mm.

10. In the system according to any one of claims 1 to 9, the at least one sample substrate holder is coupled to the at least one relative motion system and configured to enable relative motion between the at least one sample substrate holder and the at least one relative motion system.

11. In the system according to claim 10, at least one of the first and third types of reagent management units, and the at least one sample substrate holder are both coupled to the at least one relative motion system, A system configured to enable relative motion between at least one of the first and third types of reagent management units, the at least one sample substrate holder, and the at least one relative motion system.

12. The system according to claim 1, wherein the at least one microfluidic reagent applicator comprises a microfabricated chip applicator.

13. The system according to claim 1, wherein the at least one microfluidic reagent applicator comprises a droplet-on-demand actuator comprising a piezoelectric actuator.

14. The system according to claim 1, wherein the at least one microfluidic reagent applicator comprises a droplet-on-demand actuator comprising a thermal actuator.

15. The system according to claim 1, wherein the at least one microfluidic reagent applicator comprises a droplet-on-demand actuator comprising at least one piezoelectric actuator and at least one thermal actuator, and is fluidly connected to the first reagent reservoir.

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