Systems and methods for staining biological samples

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. The control system is 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 October 19, 2016, the contents of which are incorporated herein by reference.

[0002] FIELD OF THE INVENTION FIELD OF THE INVENTION

[0002] The present invention relates to systems and methods for automated staining of biological samples, and more particularly to systems and methods for accurately processing cell and tissue samples to help conserve both precious samples and reagents. [Background technology]

[0003] Three major types of automated staining equipment are currently available: dip and dunk strainers, puddle strainers, and thin-film stainers. Each of these three types of stainers is used to localize and contrast cellular structures (e.g., nuclei and cell membranes) and / or specific cellular components (e.g., proteins and nucleic acid markers) prior to examination of a biological sample (hereafter "sample") for diagnostic purposes. Typically, a series of reagents are applied to the sample to prepare it for staining and possibly for sample storage. For microscopic examination, the cell sample is typically mounted on a substrate, such as a microscope slide, and processed thereon.

[0004] "Dip-and-dunk" stainers operate by sequentially dipping microscope slides or racks of such slides into a series of reagent volumes (or baths) and are suitable for high-throughput production of laboratory slides. Control over the staining process is primarily based on the length of immersion time and the concentration of the staining reagent in the bath. However, over time, the reagent concentration in a dip-and-dunk bath changes due to reagent uptake by the sample and degradation of the reagent, which is typically left exposed to air in the bath. Furthermore, transferring reagents from one bath to another also contributes to changes in reagent concentration due to dilution and cross-contamination between reagents. Due to the difficulty in controlling reagent concentration, automated 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 critical to ensure consistency of staining between samples. In addition, antibodies for IHC and nucleic acid probes for ISH protocols are too expensive and valuable to be dispensed in large batches in the baths that dip-and-dunk stainers are characterized by. Dip-and-dunk stainers generate large amounts of waste, making them unattractive to laboratory personnel who must handle and dispose of such waste, often in accordance with strict environmental regulations.

[0005]

[0005] The paddle staining technique works by dispensing sufficient reagent onto a cell sample mounted on a horizontally positioned microscope slide, covering the sample with a "paddle" of reagent, and then leaving it to incubate for a predetermined period of time, with or without some effort to mix the reagent, such as by swirling the reagent paddle with a jet of compressed air. Once the reagent has been in contact with the sample for a predetermined amount of time, the slide is typically rinsed to remove the reagent so that new reagent can be dispensed. In some cases, slides must be washed multiple times during a particular staining protocol to ensure that the first reagent has been completely removed before a second, potentially incompatible reagent is dispensed onto the sample. Paddle technology has made it possible to automate 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 some of the reagent remains unreacted with the sample and is therefore wasted. Furthermore, when the amount of rinse aid is taken into account, the amount of waste that can be generated by a paddle stainer during a given staining protocol can be very large, leaving its disposal a burden on laboratory personnel.

[0006] "Thin-film" stainers aim to reduce reagent volume and conserve precious reagents by confining them in the capillary space between the surface of a microscope slide and a second surface, such as a cover tile or cover slip. Localized reagent uptake by the sample can lead to reagent depletion within the capillary space, which can lead to concentration gradients, which in turn can result in inconsistent staining across the sample and uncertain analysis of the staining pattern. While mixing can alleviate the staining gradient to some extent by supplying replenishment reagent to the depleted region, this approach often complicates the stainer design.

[0007] A more recent approach to conserving precious reagents involves the use of microfluidic applicators to dispense staining reagents onto small areas of a specimen. For example, Pepper et al. (Journal of Histology, 34:3, pp. 123-131, 2011) disclose the use of thermal inkjet printing for histological staining. Another example of a microfluidic applicator is disclosed by Lovchiket et al. (15th Int. Conf. on Miniaturized Systems for Chemistry and Life Sciences, Oct. 2-6, 2011, pp. 368-370, the contents of which are incorporated herein by reference) for deparaffinization.

[0008]

[0008] PCT Application No. PCT / 2016 / EP058801 discloses a system and method utilizing directional microfluidic reagent dispensing of reagent onto a sample, providing an important step toward realizing such an integrated system. This patent application is incorporated herein by reference to the extent not inconsistent with this disclosure. Briefly, a primary staining composition and a macromolecular reagent composition are provided for droplet-on-demand application. Also disclosed is a method for 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 staining reagent from the print head onto that portion of the tissue sample at a predetermined rate, which can be performed multiple times while the process is monitored. For example, by measuring the stain intensity on the sample, dispensing of the reagent can be repeated if the measured stain intensity does not reach a predetermined threshold. Dispensing can be performed with or without an overlying fluid layer. can be done. Summary of the Invention [Problem to be solved by the invention]

[0009]

[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) mounted on a substrate (such as a microscope slide), preserving tissue morphology, further conserving valuable reagents, and, in certain embodiments, making maximum use of microfluidic reagent dispensers to conserve valuable reagents while also providing more complete control over the staining process to help extract additional diagnostic information from the sample.

[0010]

[0010] Previous approaches have not addressed the delivery of less valuable bulk reagents (such as washing reagents, buffers, and deparaffinization reagents) required to prepare most or all samples for initial and subsequent delivery of staining reagents, have not adequately addressed the protection of tissue during processing, and are believed to be unsuitable for automatically preparing samples for mounting. In light of this, and as noted above, the present disclosure discloses integrated methods and systems that conserve valuable reagents and reduce waste while facilitating automation of the entire staining process. What is also needed are methods and systems that not only help preserve valuable reagents, but also better utilize valuable biological samples in obtaining additional diagnostic information. [Means for solving the problem]

[0011] In one aspect of the present disclosure, a system includes a microfluidic reagent applicator, 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 even more specific embodiments, the microfluidic reagent applicator, the bulk fluid applicator, and the fluid aspirator are combined into one unit of the system. In other even 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 be further incorporated into one unit of the system together with the fluid applicator. In an even more particular embodiment, when the bulk fluid applicator and bulk fluid aspirator are combined into one unit, the aperture of the bulk fluid applicator and the aperture of the bulk fluid aspirator are separated by a distance of at least 0.5 mm, such as a distance of at least 0.1 mm, for example, a distance of at least 1.0 mm.

[0012] In another aspect of the present disclosure, a method includes obtaining an image of a sample on a substrate, locating 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 located. In one embodiment, the method includes dispensing a bulk fluid to the location on the substrate where the sample is located and removing the bulk fluid from the location on the substrate where the sample is located. In a specific embodiment, the sample is a paraffin-embedded tissue sample, and locating the sample on the substrate includes detecting a portion of a paraffin section containing the sample and dispensing the bulk fluid substantially only to the portion of the paraffin section where the sample is located. In a more specific embodiment, the bulk fluid includes a deparaffinizing reagent. In this manner, wells can be formed in the non-polar paraffin surrounding the sample and can be used to retain 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 the steps of selecting two or more distinct portions of the site on the substrate where the sample is located, and dispensing a deparaffinizing fluid using a bulk fluid applicator onto the two or more selected distinct portions of the site where the sample is located to produce two or more wells in the paraffin located on the two or more selected distinct portions of the site where the sample is located. In an even more specific embodiment, the method further includes dispensing the deparaffinizing fluid onto a selected one of the two or more distinct portions of the site where the sample is located, and simultaneously removing the deparaffinizing fluid from the sample using a fluid aspirator. Advantageously, the bulk fluid applicator and the fluid aspirator are combined into one unit and move together to simultaneously dispense and remove the deparaffinizing fluid, thereby rapidly removing the paraffin from the selected portions of the sample.

[0014] In certain embodiments, the separation distance between the bulk fluid applicator aperture and the fluid aspirator aperture can be at least 1.0 mm or greater (up to 10 mm, about 20 mm, about 30 mm, or greater, e.g., up to about 100 mm, about 200 mm, about 300 mm, or even up to about 1 cm) while maintaining a connecting fluid flow between the applicator aperture and the aspirator aperture. In certain embodiments, the bulk fluid applicator and fluid aspirator can be a pair of needles spaced apart from each other (e.g., a pair of needles spaced apart 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 manner, for example, a fluid knife can be moved across the sample to selectively deparaffinize all or a portion of a paraffin-embedded tissue sample. In more specific embodiments, such fluid knives can be used to prepare generally square or rectangular wells over selected portions of a sample, into which further reagents can be deposited and removed according to a staining protocol. In even more specific embodiments, a pair of needles can be rotated about a central axis to form a rotating fluid knife, which can be used to prepare circular wells over selected portions of a sample when used to dispense and remove deparaffinization fluid. In either case, individual wells can be formed over selected portions of a sample, and different diagnostic assays can be performed on a single sample in these separate wells, thereby obtaining additional diagnostic information from a single valuable resource.

[0015] In another aspect of the system of the present disclosure, at least two adjacent microfluidic reagent dispenser ports of a single 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 or thermal inkjet printer head, alternating rows or alternating columns of the matrix are fluidly connected to at least two separate reagent reservoirs of the microfluidic reagent dispenser. In an alternative embodiment, alternating microfluidic dispenser ports within one or more rows or columns of a matrix of microfluidic dispenser ports of a piezoelectric or thermal inkjet printer head may be in fluid communication with at least two separate reagent reservoirs of the microfluidic dispenser. In some embodiments, at least two or more different subsections of the matrix of microfluidic dispenser ports of a piezoelectric or thermal inkjet printer head can be fluidly connected to at least two or more separate reagent reservoirs. In other embodiments, valves can control which reagents are sequentially delivered to the matrix of microfluidic dispenser ports according to a given staining protocol, especially if the reagents are compatible with each other (such as primary, secondary, and detection system antibodies and reagents).

[0016] In another aspect of the present disclosure, a method involves sequentially or simultaneously depositing at least two staining reagents onto a tissue sample at substantially the same location so that the at least two staining reagents contact the sample simultaneously. Even reagents that are not normally compatible can be dispensed onto the sample simultaneously or in rapid succession from separate microfluidic reagent dispensers (or separate microfluidic dispenser ports on a single microfluidic reagent dispenser). For example, hematoxylin and eosin (H&E) can be deposited onto the sample together, significantly reducing the time required to prepare an H&E-stained sample.

[0017]

[0017] Another aspect of the present disclosure is a non-transitory computer-readable medium for automated processing of at least a portion of a sample held on a substrate, wherein a memory includes (a) instructions for obtaining an image of the sample on the substrate, (b) instructions for automatically locating the location of the sample on the substrate, and (c) instructions for dispensing a fluid at the location of the sample on the substrate.

[0018] In summary, the disclosed systems and methods represent improvements to the development, quality, and patient-safe processes involved within the histology tissue staining industry. In certain embodiments, a reagent deposition device is configured to allow any dispensed reagent to penetrate a thin boundary layer of fluid and replenish the 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 puddles of staining reagent that passively diffuse a concentration gradient within the tissue sample. In these staining systems, which are believed to lack active mixing of reagent at the puddle-tissue interface, the diffusion of stain into the tissue is affected by the buildup of a stain concentration depletion layer at the interface, limiting staining kinetics. It is believed that the present disclosure provides an improvement over prior art staining techniques by overcoming the limitations of passive stain diffusion by (i) creating a stain film with a thickness approximating the thickness of the depletion layer and (ii) replenishing stain molecules at the depletion layer.

[0019]

[0019] Further features and advantages of the disclosed systems and methods will become apparent from a consideration of the following detailed description, taken in conjunction with the accompanying drawings. [Brief explanation of the drawings]

[0020] [Figure 1] FIG. 1 is a schematic diagram of an embodiment of the disclosed system. [Figure 2] FIG. 2 is a schematic diagram of another embodiment of the disclosed system including a conveyor and multiple modules for processing samples according to staining protocols. [Figure 3] FIG. 3 is a schematic diagram of another embodiment of the disclosed system including a stationary sample substrate holder and an array of movable sample processing modules. [Figure 4] Figure 4A illustrates a first embodiment of a combined bulk fluid applicator and fluid aspirator module, and Figure 4B illustrates a second embodiment of a combined bulk fluid applicator and fluid aspirator module further including an air knife. [Figure 5] Figure 5A is a diagram of an embodiment of a combined bulk fluid applicator and fluid aspirator module configured to prepare square or rectangular wells in the paraffin of a paraffin-embedded tissue section, and Figure 5B is a diagram of an embodiment of a combined bulk fluid applicator and fluid aspirator module configured to prepare circular wells in the paraffin of a paraffin-embedded tissue section. [Figure 6] Figure 6A illustrates an embodiment of a droplet-on-demand microfluidic reagent applicator with an integrated reagent reservoir, Figure 6B illustrates an embodiment of a droplet-on-demand microfluidic reagent applicator with a replaceable reagent reservoir, Figure 6C illustrates an embodiment of a droplet-on-demand microfluidic reagent applicator with a remote reagent reservoir, and Figure 6D illustrates an embodiment of a droplet-on-demand microfluidic reagent applicator with a remote reagent reservoir and an integrated intermediate reservoir. [Figure 7] FIG. 7 is a schematic diagram illustrating how reagent reservoirs can be kept cool when not in use according to certain embodiments of the disclosed system. [Figure 8] FIG. 8 is a schematic diagram illustrating how multiple reagent reservoirs can be used with a single droplet-on-demand actuator head according to certain embodiments of the disclosed system. [Figure 9]Figure 9A is a schematic diagram showing the front view of an embodiment of an integrated system including a microfluidic droplet-on-demand actuator, a bulk fluid applicator slit, and a fluid aspirator slit, Figure 9B is a schematic diagram showing the side view of an embodiment of an integrated system including a microfluidic droplet-on-demand actuator, a bulk fluid applicator slit, and a fluid aspirator slit, and Figure 9C is a schematic diagram showing the bottom view of an embodiment of an integrated system including a microfluidic droplet-on-demand actuator, a bulk fluid applicator slit, and a fluid aspirator slit. [Figure 10] Figure 10A is a schematic diagram showing the front, side, and bottom views of an embodiment of an integrated system including a microfluidic droplet-on-demand actuator, a bulk fluid applicator needle, and a fluid aspirator needle. Figure 10B is a schematic diagram showing the side, and Figure 10C is a schematic diagram showing the bottom, of an embodiment of an integrated system including a microfluidic droplet-on-demand actuator, a bulk fluid applicator needle, and a fluid aspirator needle. [Figure 11] FIG. 11 is a schematic side view of an embodiment of a subsystem in the disclosed system that includes a microfluidic reagent applicator and a combined bulk fluid applicator and fluid aspirator mechanism configured for waste separation. [Figure 12] FIG. 12 is a schematic top view of an embodiment of a subsystem including a microfluidic reagent applicator and a combined bulk fluid applicator and fluid aspirator mechanism in the disclosed system. [Figure 13] FIG. 13 is a schematic top view of an embodiment of a subsystem including a microfluidic reagent applicator and a combined bulk fluid applicator / fluid aspirator / air knife mechanism in the disclosed system. [Figure 14]FIG. 14 is a schematic front view of an embodiment of a subsystem in the disclosed system that includes a sample substrate holder configured to sealingly mate with a microfluidic reagent applicator to form a chamber in which samples can be treated at elevated temperatures and / or pressures. [Figure 15] Figures 15A and 15B show two deparaffinization methods. [Figure 16] FIG. 16 shows a schematic diagram demonstrating the overall functional flow chart for controlling the printing operation in an ink jet printer. [Figure 17] FIG. 17 is a schematic diagram showing an embodiment of a control method for an inkjet dyeing system. DETAILED DESCRIPTION OF THE INVENTION

[0021]

[0046] As used herein, the singular terms "a," "an," and "the" are intended 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," and the like are used interchangeably and are intended to have the same meaning. Similarly, the terms "comprises," "includes," "has," and the like are used interchangeably and are intended to have the same meaning. Specifically, each of these terms is defined consistent with the general U.S. patent law definition of "comprising" and, therefore, is intended to be open-ended, meaning "at least the following," and further, is intended to be interpreted as not excluding additional features, limitations, aspects, and the like. Thus, for example, "a device having components a, b, and c" means that the device includes at least components a, b, and c. Similarly, the phrase "a method 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 a particular order herein, those skilled in the art will recognize that the order of the steps and processes may vary unless a specific order is clearly indicated by the context.

[0023]

[0048] As used herein, the term "about" refers to ±1 to 10% of the cited numerical value, for example, ±1 to 5% of the cited numerical value, such as ±1 to 2% of the cited numerical value.

[0024]

[0049] As used herein, the term "substantially" refers to at least 90%, for example, at least 95%, such as at least 99% of the object referred to by this term.

[0025]

[0050] As used herein, the term "antibody" refers to an immunoglobulin or immunoglobulin-like molecule, including, by way of example and not limitation, IgA, IgD, IgE, IgG, and IgM, combinations thereof, and similar molecules produced during the immune response of any vertebrate (e.g., in mammals such as humans, goats, rabbits, and mice), which specifically bind to a molecule of interest (or a group of closely related molecules of interest) to 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 sFv fragments, and dsFv fragments), and the like. Included are dsFv fragments, F(ab)'2 fragments, single-chain Fv proteins ("scFv"), disulfide-stabilized Fv proteins ("dsFv"), diabodies, triabodies, and camelid antibodies. Antibody further refers to a polypeptide ligand comprising at least a light or heavy chain immunoglobulin variable region that specifically recognizes and binds to an epitope of an antigen. An antibody may be composed of a heavy chain and a light chain, each of which has a variable region, termed a variable heavy chain (VH) region and a variable light chain (VL) region. The VH and VL regions together are responsible for binding to the 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 capable of being specifically bound by a 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 macromolecules such as, for example, haptens, simple intermediary metabolites, sugars (e.g., oligosaccharides), lipids, and hormones, as well as complex carbohydrates (e.g., polysaccharides), phospholipids, nucleic acids, and proteins.

[0027]

[0052] As used herein, the terms "biological sample" or "sample" refer to any solid or fluid sample obtained from, excreted by, or secreted by any living organism, including, but not limited to, unicellular organisms such as bacteria, yeast, protozoa, and amoebas, multicellular organisms (such as plants or animals, including samples from healthy or apparently healthy human subjects, or human patients suffering from a condition or disease to be diagnosed or studied, such as cancer), among others. Specifically, the sample can be one that is 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, the biological sample can be, for example, blood, plasma, serum, urine, bile, ascites, saliva, cerebrospinal fluid, aqueous humor, or vitreous humor, or any bodily secretion, transudate, or exudate (e.g., fluid obtained from an abscess or any other site of infection or inflammation), or fluid obtained from a joint (e.g., a normal joint or a diseased joint). A biological sample may also be a sample obtained from any organ or tissue (including a biopsy or autopsy specimen such as a tumor biopsy), or may include cells (either primary or cultured cells), or medium conditioned by any cell, tissue, or organ. In some examples, the biological sample is a nuclear extract. In particular examples, the sample is a quality control sample. In particular examples, the sample is a test sample. For example, the test sample is 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 for or suffering from a disease. The sample can be prepared using any method known to those of ordinary skill in the art. The sample can be obtained from a subject for routine screening or from a subject suspected of having a disorder such as a genetic abnormality, infection, or neoplasia. Additionally, embodiments of the disclosed methods can be applied to samples that do not have a genetic abnormality, disease, disorder, etc., referred to as "normal" samples. The sample can include multiple targets that can be specifically bound by one or more detection probes.In particular examples, the sample is a tissue section excised from a block of paraffin-embedded tissue that has been mounted on (and possibly baked on) a microscope slide. In other particular examples, the sample is a cytology or hematology sample that has been prepared by depositing cells onto a microscope slide (such as by contacting a filter on which the cells have been 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) refer to staining techniques that deposit discrete droplets of a reagent onto a target sample, as opposed to "flooding" a slide or sample thereon with the reagent. In certain embodiments, droplet-on-demand techniques utilize inkjet or piezoelectric technology. In some embodiments disclosed herein, droplet dispensing techniques are facilitated using inkjet printheads or similar technology.

[0029]

[0054] As used herein, the term "humectant" refers to a hygroscopic substance used to keep a substance, such as a tissue sample, moist, as opposed to a desiccant. It is often a molecule containing various hydrophilic groups, most often hydroxyl groups, although amine and carboxyl groups, sometimes esterified, may also be encountered (the affinity for forming hydrogen bonds with water molecules is an essential attribute). Humectants are thought to attract and retain moisture from nearby air by absorption, drawing water vapor into and / or beneath the surface of the organism / subject. In contrast, desiccants also attract ambient moisture, but adsorb rather than absorb, the water vapor by condensing on the surface as a coating layer. In the context of inkjet deposition or similar techniques, humectants are plausibly important for maintaining viable nozzles. In certain embodiments, it is important to keep tissue or biological samples hydrated during thin-film processing.

[0030]

[0055] The term "inkjet" in this disclosure refers to a family of drop-on-demand technologies that use piezoelectric (or thermal) elements to actuate droplets from a distribution manifold, which may include direct and non-contact methods common to 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 can be detected 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" for detecting a target in a tissue sample.

[0033]

[0058] As used herein, the term "primary stain" refers to a dye or similar molecule that enhances contrast in a tissue sample. In certain embodiments, a primary stain directly "labels" biological structures within or on cells without the use of a specific binding agent such as an antibody. 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 safranin. Other examples of primary stains include stains used to stain bacteria (Gram-positive or Gram-negative stains), stains used to identify endospores (endospore stains), stains used to aid in the identification of Mycobacterium tuberculosis species (Ziehl-Neelsen stains), Papanicolaou stain kits (using a combination of hematoxylin, orange G, eosin Y, light green SF yellowish, and sometimes Bismarck brown Y), periodic acid-Schiff stains ("PAS stains"), silver stains, etc. Still other non-limiting primary stains include (i) histological stains for selectively showing Mycobacterium and other acid-fast organisms or components (e.g., AFB, available from Ventana Medical Systems Inc. (hereinafter Ventana, Tucson, Arizona, USA)).III staining kit), (ii) histological stains to distinguish acidic mucins from neutral polysaccharides (e.g., Alcian Blue for PAS, also available from Ventana), (iii) histological stains to demonstrate weakly acidic mucopolysaccharides (e.g., Alcian Blue staining kit, also available from Ventana), (iv) histological stains for Helicobacter pylori (e.g., Alcian Yellow staining kit, also available from Ventana), (v) histological stains to selectively demonstrate amyloid (e.g., Congo Red staining kit, also available from Ventana), (vi) histological stains to distinguish acidic mucins from neutral polysaccharides (e.g., Dimethicone Red staining kit, also available from Ventana), (vii) histological stains for showing elastic fibers in tissue sections (e.g., Elastic Stain Kit, also available from Ventana); (viii) histological stains for differentiating leukocytes in bone marrow and other hematopoietic tissues (lymph nodes) (e.g., Giemsa Stain Kit, also available from Ventana); (ix) histological stains for showing polysaccharides in the cell walls of fungi and other opportunistic infectious organisms, including, but not limited to, stains capable of distinguishing pathogenic fungi such as Aspergillus and Blastomyces, and other opportunistic infectious organisms such as Pneumocystis carinii (e.g., Giemsa Stain Kit, also available from Ventana).II staining kit), (x) histological stains for showing gram-negative and gram-positive bacteria (e.g., Gram stain kits also available from Ventana), (xi) histological stains used to study connective tissue, muscle, and collagen fibers (e.g., Trichrome Green, also available from Ventana), (xii) histological stains for detecting iron pigments in bone marrow, hemochromatosis tissue, and hemosiderosis (e.g., Iron Staining Kits also available from Ventana), (xiii) histological stains for showing capillary basement membranes (e.g., Jones H&E Staining Kit or Jones Light Green Staining Kit, both also available from Ventana), (xiv) histological stains for detecting fungi (e.g., (xv) histological stains for detecting acidic mucopolysaccharides (mucins) (e.g., Muciarmine stain kit also available from Ventana); (xvi) histological stains used to demonstrate the presence of glycogen, including stains that can aid in the identification of positive reticular fibers, basement membrane, fungi, and neutral mucopolysaccharides, or stains that can aid in distinguishing PAS-positive secretory adenocarcinoma from undifferentiated PAS-negative squamous cell carcinoma (e.g., PAS stain kit also available from Ventana); (xvii) histological stains for demonstrating reticular fibers (e.g., Reticulum II stain kit also available from Ventana); (xviii) histological stains used to study certain argyrophilic microorganisms (e.g., Steiner II stain kit also available from Ventana); (xix) histological stains used to detect certain gastric ulcers (H. pylori (H.(xx) histological stains for studying connective tissue, muscle, and collagen fibers (e.g., Trichrome II Blue Stain Kit, also available from Ventana); and (xxi) histological stains for studying connective tissue, muscle, and collagen fibers (e.g., Trichrome III Blue Stain Kit, or Trichrome III Green Stain Kit, each also available from Ventana). Those skilled in the art will also recognize that there are other primary stains, or dyes for that matter, that can be used in conjunction with the kits, methods, and compositions (e.g., primary staining compositions, reagent compositions) of the present disclosure.

[0034]

[0059] As used herein, the term "reagent" may refer to any fluid deposited on a tissue section or cytology sample used in the context of morphological (e.g., hematoxylin and eosin), immunohistochemical, or special staining. This includes, but is not limited to, oils, organics, and cross-linking reagents to remove wax (i.e., deparaffinization), washes, rinses, diluents, or buffers used to set reaction conditions, diluting reagents to achieve appropriate concentrations, stop reactions, or wash 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 used in IHC or ICC staining.

[0035]

[0060] As used herein, "surfactants" are classified as anionic, cationic, or nonionic, depending on their mode of chemical action. Generally, surfactants reduce 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 tail faces inward and the polar or ionic head faces outward toward the aqueous environment. The nonpolar tail creates a nonpolar "pocket" within the micelle. Nonpolar compounds in solution are sequestered in the pocket formed by the surfactant molecules, thus allowing the nonpolar compounds to remain mixed in the aqueous solution. In certain embodiments, surfactants can be used to produce uniform diffusion of reagents across tissue sections while also reducing background staining.

[0036]

[0061] As used herein, a "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. Further examples of targets typically include proteins, peptides, nucleic acids, sugars, and lipids. Reagents for use in the present disclosure may be capable of converting a target substance present in a biological sample into a detectable form so that the location of the target can be detected (such as visually).

[0037]

[0062] One aspect of the present disclosure is an automated biological sample staining system including 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 some 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), the microfluidic reagent applicator(s), the bulk fluid applicator(s), and the fluid aspirator(s), together or separately, in any combination. In some embodiments, the automated biological sample staining system further includes a control system programmed to execute at least one staining protocol on a sample mounted on a substrate held in the sample substrate holder. In some embodiments, the system includes two or more microfluidic reagent applicators.

[0038]

[0063] In some embodiments, 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 execute individual steps of at least one staining protocol. In some embodiments, the control system may also control one or more auxiliary subsystems that operate in combination with these components to process samples according to a particular staining protocol. The number of staining protocols stored in the control system's memory (e.g., non-transitory memory) and the number of instructions and parameters measured and / or applied to execute individual steps of a particular staining protocol are not limited but are typically scaled according to the overall system complexity. That is, in an embodiment for staining individual slides according to one particular staining protocol (e.g., a small-scale system for rapid H&E staining of frozen tissue sections in an operating room), the number of instructions and parameters stored in the control system memory may be minimized. However, in a complex system that receives a variety of different types of specimens that are processed 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 needed to reliably and repeatedly perform any number of staining protocols.

[0039]

[0064] Examples of additional subsystems that may be in communication with and controlled by the control system and / or 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 may be cooled), one or more reagent transfer systems, and one or more substrate transfer systems. Further examples of additional subsystems that may be in communication with and under the control of the control system are 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 fluid movement from a sample using compressed gas, such as compressed air or nitrogen. For example, instead of aspirating fluids from a sample and directing them to waste, the fluids can be moved or "blown off" into a waste container or a waste receiver leading to a waste container.

[0041]

[0066] In some embodiments, various system components (e.g., those identified in FIG. 1 ) may be combined to form one or more reagent management units, each having the same or different configurations (e.g., configurations refer to the type of system component, the number of system components, or the quality of any one system component). In light of this, the systems disclosed herein may include 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 certain embodiments, at least one bulk fluid applicator and at least one fluid aspirator are combined with at least one first-type 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 with at least one second-type 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 with at least one third-type 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 with at least one fourth-type reagent management unit. Depending on the system configuration, the system may include any combination of two or more of the first, second, third, and fourth-type reagent management units. In some embodiments, the reagent management unit can include, for example, pumps, reservoirs, valves, etc., and a controller for controlling these to deliver predetermined volumes of predetermined fluids. In some embodiments, the reagent management unit can also include other means for delivering one or more reagents, which can be solid or liquid. For example, the reagent management unit can include a reconstitution unit for dissolving a solid reagent into a liquid for dispensing into the sample. Alternatively, the reagent management unit can also include one or more single-dose reagent applicators, such as a blister pack and associated mechanisms for managing and dispensing the contents of the blister for contact with the sample.

[0043]

[0068] In certain embodiments, the at least one bulk fluid applicator and the at least one fluid aspirator comprise a pair of needles, and in yet another particular example, the pair of needles are separated by a distance of at least 0.5 mm, such as a distance of at least 0.1 mm, e.g., a distance of at least 1.0 mm.

[0044]

[0069] Regardless of the type of the at least one reagent management unit (i.e., the selection and / or number of system components), the reagent management unit can be coupled to at least one relative motion system, or the 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 can be 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 to provide relative motion between the respective components.

[0045]

[0070] In certain embodiments, 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, e.g., up to about 1 cm, 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, such as those described above, the bulk fluid applicator and bulk fluid aspirator can be a pair of needles separated from each other (e.g., by about 1 mm to about 100 mm, such as 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."

[0046]

[0071] For example, and in certain embodiments, a fluid knife can be moved across a paraffin-embedded tissue sample by relative motion to selectively deparaffinize all or a portion of the sample. In more specific embodiments, such a fluid knife can be used to prepare 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 even more specific embodiments, a pair of needles can be rotated about a central axis to form a rotating liquid knife, which, when used to dispense and remove deparaffinization fluid, can be used to prepare a circular well over a selected portion of the sample.

[0047]

[0072] In certain embodiments of the disclosed systems, at least one microfluidic reagent applicator comprises at least one microfabricated chip applicator, such as that disclosed in Lovchik et al. (15th Int. Conf. on Miniaturized Systems for Chemistry and Life Science, Oct. 2-6, 201, pp. 368-370), the contents of which are incorporated herein by reference. In other specific 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 even more specific embodiments, the disclosed systems can include any combination of two or more of a microfabricated tip applicator, a piezoelectric applicator, and a thermal applicator. For example, a piezoelectric actuator may be selected to dispense fluids containing reagents susceptible to sheer force degradation or thermally unstable reagents, while a microfabricated tip applicator or thermal actuator may be selected to dispense fluids containing reagents not susceptible to sheer force degradation or thermally unstable reagents, respectively. Degradation of a particular reagent can be determined by comparison with the staining performance of that particular reagent on a paddle or with a thin film staining system as described in the background above.

[0048]

[0073] In other specific embodiments, the microfluidic reagent applicator includes an integrated reagent reservoir, such as one that is fluidly connected to the droplet-on-demand actuator. In certain embodiments, the microfluidic reagent applicator includes a remote reagent reservoir, e.g., a remote reagent reservoir that is fluidly connected to the droplet-on-demand actuator. In yet other specific embodiments, the microfluidic reagent applicator can include a replaceable reservoir. The replaceable reservoir is fluidly connected to the microfluidic reagent applicator upon coupling of the droplet-on-demand actuator and the replaceable reagent reservoir that is fluidly connected to the droplet-on-demand actuator upon coupling of the droplet-on-demand actuator and the replaceable reagent reservoir. The microfluidic reagent applicator can also include an intermediate reagent reservoir. The intermediate reagent reservoir is fluidly connected to and fed by the remote reagent reservoir. For example, in a more specific embodiment, the microfluidic reagent applicator includes 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 recognize that the disclosed systems can include any combination of reservoir configurations and / or bulk fluid applicators as described herein. For example, valuable primary antibodies and nucleic acid probes can be provided in a microfluidic applicator with integrated reservoirs, which can be moved in and out of a cooled reagent storage system and delivered to a specific sample substrate holder as needed for a specific staining protocol. Alternatively, such valuable reagents can be held in replaceable reservoirs, which can be moved in and out of a cooled reagent storage system and coupled to a microfluidic reagent applicator such as a droplet-on-demand actuator. Meanwhile, less valuable reagents, such as bulk fluids containing wash solutions, deparaffinization fluids, etc., can be held in remote, easily refillable reagent reservoirs and fluidly connected to the microfluidic reagent applicator or bulk fluid applicator (through tubing and possibly via intermediate reservoirs). 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 reservoirs adjacent 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 possibly intermediate reservoirs). In certain embodiments, all different reagents used for a particular detection chemistry (e.g., di-aminobenzidine detection of primary antibody binding) are held in reservoirs fluidly connected to a single microfluidic reagent applicator (either directly, through tubing, or via tubing and intermediate reservoirs) and directed sequentially or simultaneously through a single microfluidic reagent applicator in any combination onto the sample. In even more specific embodiments, at least two adjacent microfluidic dispenser ports of a single microfluidic reagent dispenser are fluidly connected to at least two separate reagent reservoirs of the microfluidic reagent dispenser.As previously described, for example, in a matrix of microfluidic dispenser ports in a piezoelectric or thermal ink jet printer head, alternating rows or alternating columns of the matrix fluidly connect to at least two separate reagent reservoirs of a microfluidic reagent dispenser. In other alternative embodiments, alternating microfluidic dispenser ports in one or more rows or columns of a matrix of microfluidic dispenser ports in a piezoelectric or thermal ink jet printer head fluidly connect to at least two separate reagent reservoirs of the microfluidic dispenser. In certain embodiments, at least two or more different subsections of a matrix of microfluidic dispenser ports in a piezoelectric or thermal ink jet printer head fluidly connect to at least two or more separate reagent reservoirs. In yet other embodiments, valves can control which reagents are sequentially delivered to a matrix of microfluidic dispenser ports according to a given staining protocol, especially when the reagents are compatible with each other (such as primary, secondary, and detection system antibodies and reagents).

[0050]

[0075] Also disclosed herein is a method for automatically processing at least a portion of a sample held on a substrate. The method includes obtaining an image of the sample on the substrate, automatically determining the location of the sample on the substrate, and dispensing a fluid at the location of the sample on the substrate. In one embodiment, dispensing the fluid at the location of the sample on the substrate includes dispensing the fluid substantially only at the location of the sample on the substrate. In certain embodiments, the method can further include removing the fluid from the site on the substrate where the sample is located. In more specific embodiments, the sample includes a paraffin-embedded sample, and determining the location of the sample on the substrate includes automatically detecting a portion of a paraffin section containing the sample. In even more specific embodiments, the fluid includes a deparaffinization fluid, and dispensing the fluid can include dispensing the deparaffinization fluid onto at least one sub-portion of the paraffin section containing a sub-portion of the sample, and can further include removing the deparaffinization fluid from at least one sub-portion of the paraffin section containing the sub-portion of the sample to form a well in the paraffin around the sub-portion of the sample. Alternatively, the fluid may comprise a deparaffinizing fluid, and the step of pouring the fluid may comprise pouring the deparaffinizing fluid onto substantially the portion of the paraffin section containing the sample, and further comprising removing the deparaffinizing liquid from the substantial portion of the paraffin section containing the sample, leaving the paraffin on the substrate around the sample to form a well in the paraffin substantially surrounding the sample. The pouring of the deparaffinizing fluid and the removal of the deparaffinizing fluid may occur simultaneously in some embodiments.

[0051]

[0076] Once the well (or wells) is formed around the sample (or one or more subportions of the sample), the method can further include dispensing at least one second fluid into the well (or wells) within the paraffin. While not wishing to be bound by any particular theory, it is believed that wells within the paraffin can benefit from the advantage of confining polar (such as aqueous) solutions within the wells, as the hydrophobic paraffin forms a barrier to migration of polar solutions. For example, the second fluid can be one or more of water, a buffer, an antibody solution, a dye solution, a nucleic acid solution, a solvent, a surfactant, and a humectant, which enters the wells within the paraffin.

[0052]

[0077] In more specific embodiments, the disclosed method includes selecting two or more distinct sample subportions from a sample location on a substrate and dispensing a deparaffinizing fluid onto the selected two or more distinct sample subportions to dissolve the paraffin on the selected two or more distinct sample subportions. In certain embodiments, the method further includes removing the deparaffinizing fluid from the two or more distinct sample subportions to form two or more distinct wells in the paraffin surrounding the two or more distinct sample subportions. In even more specific embodiments, the disclosed method includes selecting two or more distinct subportions from images of H&E-stained serial sections of the same sample block from which the samples were obtained. Because H&E-stained serial sections (meaning sections sliced ​​by a microtome or other means from one of several samples from which two or more distinct subportions have been selected) have roughly the same overall shape as the sample from which the two or more distinct subportions were selected, specific morphological features identified in images of the H&E-stained serial sections can be identified and mapped to similar subportions of the sample (using image analysis techniques well known in the art) and used to guide further processing of the sample. For example, two or more distinct subportions of the sample mapped from the serial sections are selected to match different morphological features of the sample. In one embodiment, two or more distinct subportions of the sample mapped from the serial H&E sections are selected to serve as at least one positive control or negative control, and to provide at least one subportion of the sample for comparison with at least one of the positive control and negative control.

[0053]

[0078] In another embodiment, 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 may be considered incompatible with each other, such as hematoxylin and eosin, can be advantageously deposited together onto the sample from one or more microfluidic reagent dispensers, e.g., from adjacent microfluidic dispenser ports or separate microfluidic reagent dispensers.

[0054]

[0079] In some embodiments, real-time dispensed volume measurement data may be stored and matched with slide specimen identifiers, and each dispenser identifier may be associated with the delivery of reagent to the specimen. This metadata may be stored on the instrument or on a host computer for tracking and reporting purposes in histology studies. Dispensed volume metadata may be tracked for the entire slide staining process history. In addition, continuous performance tracking may be matched for each dispenser identifier throughout its lifetime. For a given "poor dispense," the "failed" dispenser and affected specimen may be flagged by software, for example, and reported to the histologist via various electronic methods (i.e., LED indicator, run report, etc.) to enhance patient safety. Dispensed volume metadata may be collected in an external data bank for research and development purposes. This data can be used to qualify and screen new staining kits or individual staining products. Additionally, dispensing verification tracking can also be used on newly developed reagents, as these may perform differently over time and may further affect the dispensing delivery of the reagent to the specimen slide (i.e., material compatibility with the reagent and dispenser).

[0055]

[0080] In some embodiments, a reagent, or a composition containing the reagent, is dispensed from a microfluidic reagent dispenser through an immiscible fluid at a sufficient rate to drive droplets of the reagent through a thin film of tissue-preserving fluid medium. Examples of thin film fluids include, but are not limited to, draksol, Lympar, mineral oil, or silicone oil. Generally, favorable attributes include a liquid state at room temperature (e.g., 20-30°C), low surface tension, and low vapor pressure. In some embodiments, an immiscible barrier layer may allow aqueous liquids to be resupplied through the barrier to the sample surface. The low surface tension allows the barrier to be coated onto the sample as a relatively thin film (approximately 100 μm or less in height). Furthermore, the low vapor pressure may ensure that the barrier layer evaporates slowly from the sample. This may drive the reagent into a layer below the immiscible fluid that is in communication with the sample. In this embodiment, the kinetic energy of the droplet (the product of the mass of the droplet and the impact velocity at which the droplet strikes the membrane) must be greater than the surface tension / energy of the protective layer (plus provide enough additional energy to account for the displaced fluid), e.g., about 9.52x10 -10 In one embodiment, the kinetic energy is greater than about 6.23×10 -10 J. Furthermore, to ensure that droplet breakage does not occur upon impact, the Weber number of the droplet must be less than about 18. In some embodiments, the droplet must have a higher density than the protective layer to ensure that once the surface breaks, the droplet remains in direct contact with the sample through the protective layer.

[0056]

[0081] In other embodiments, reagents are dispensed into a pre-existing fluid "layer" at a velocity sufficient to drive reagent droplets into a thin film that locally transports the stain through the layer to a fluid-tissue stain-depleted layer. This is believed to facilitate replenishment of reagent at the interface contact point with the sample. This, in turn, is believed to eliminate the stain-depleted boundary layer and improve staining reaction kinetics, which are sometimes affected by diffusion of the staining reagent across the depleted layer. Indeed, for large biomolecules such as antibodies, binding of the molecule to its target is time- and concentration-determining. Dispensing by the devices disclosed herein (and the associated inherent mixing) is believed to enhance effective concentration at the tissue surface by continuously disrupting the thin film with additional reagent material, thereby increasing uptake rates. In this embodiment, velocities are typically in the range of about 5 m / s to about 15 m / s.

[0057]

[0082] One embodiment of an automated vital staining system disclosed in accordance with certain embodiments of the present disclosure is shown in FIG. 1. System 100 includes a control system 102. Control system 102 communicates with various subsystems and may communicate with a network 104. Network 104 may also communicate with control systems for additional automated vital staining systems, a pathology laboratory workflow control and tracking system, and a laboratory information system (LIS) and / or a hospital information system (HIS). Orders for particular samples prepared in the pathology laboratory may be sent to control system 102 and stored in the control system's memory (not shown) until the samples reach system 100. For example, a particular sample placed on a microscope slide may be guided toward system 100 by a substrate transport / substrate movement system 106 (which may be a 1-D or 2-D transport system, such as a conveyor belt or a magnetic transport system). When a sample arrives in the system 100 (either manually or automatically), the substrate identification system 108 identifies the sample based on an identifier (such as a unique sample identifier) ​​associated with the microscope slide (e.g., a barcode label, a numeric identifier, or an RFID tag), and the control system 102 associates this particular sample with instructions specifying processing steps (which can be stored in the control system's 102 memory or sent to the control system over the network 104 along with the instructions). An image acquisition system 110 can also function as part of the substrate identification system 108, capturing images of the sample (e.g., a paraffin-embedded tissue sample) and generating 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 a GUI 114, and a user can interact with the control system 102 (such as via a touch screen) to control movement and / or processing steps. The GUI 114 can also be used, for example, to monitor the progress of samples processed by the system 100, display alerts, and perform quality control checks.

[0058]

[0083] The control system 102 may include known components such as a processor, an operating system, system memory, memory storage devices, input / output controllers, input / output devices, and display devices. It may also include cache memory, data backup units, and many other devices. Examples of input devices include keyboards, cursor control devices (e.g., mice), microphones, scanners, etc. Examples of output devices include display devices (e.g., monitors or projectors such as the GUI 114), speakers, printers, network cards, etc. Display devices may include devices that provide visual information, which may typically be logically and / or physically organized as an array of pixels. An interface controller may also be included, which may include any of a variety of known or future software programs for providing input and output interfaces. Typically, the interface is operable to accept user input using selection or input means known to those skilled in the relevant art. The interface may also be a touchscreen device. In the same or alternative embodiments, an application on a computer may employ an interface that includes what is called a "command line interface" (often referred to as a CLI). A CLI typically provides text-based interaction between the application and the user. Typically, a command line interface presents output through a display device and accepts input as lines of text. For example, some embodiments may include what is called a "shell," such as Unix Shells, known to those skilled in the relevant art, or Microsoft Windows Powershell, which employs an object-oriented programming architecture, such as the Microsoft .NET framework.

[0059]

[0084] Those skilled in the relevant art will recognize that the interface may include one or more GUIs, CLIs, or a combination thereof. The processor may include a commercially available processor, such as a Celeron, Core, or Pentium processor manufactured by Intel Corporation, a SPARC processor manufactured by Sun Microsystems, or an Athlon, Sempron, Phenom, or Opteron processor manufactured by AMD Corporation, or any other processor currently available or hereafter available. Some processor embodiments may include what are known as multi-core processors and / or may employ parallel processing techniques in single or multi-core configurations. For example, multi-core architectures typically include two or more processor "execution cores." In this example, each execution core may execute as an independent processor, enabling the concurrent execution of multiple threads. Additionally, those skilled in the relevant art will recognize that the processor may be configured with what are commonly referred to as 32- or 64-bit architectures, or other architectural configurations now known or that may be developed in the future.

[0060]

[0085] The processor typically runs an operating system. For example, the operating system may be a Windows-type operating system from Microsoft Corporation, the Mac OSX® operating system from Apple Computer Corp., a Unix- or Linux-type operating system available from a number of vendors, or what are known as open source, other, or future operating systems, or some combination of these. The operating system interfaces with firmware and hardware in well-known ways and assists the processor in coordinating and executing the functions of various computer programs, which may be written in a variety of programming languages. The operating system typically cooperates with the processor to coordinate and execute the functions of the other components of the computer. The operating system also provides scheduling, input / output control, file and data management, memory management, and communication control, and related services, all in accordance with known techniques.

[0061]

[0086] System memory can be used to store desired information and can include any of a variety of known or future memory storage devices that can be accessed by a computer. Computer-readable storage media can include volatile and nonvolatile, removable and non-removable media, implemented in any method or technology for storage of information such as computer-readable instructions, data structures, program modules, or other data. Examples include any commonly available random access memory (RAM), read-only memory (ROM), electronically erasable programmable read-only memory (EEPROM), digital versatile disks (DVDs), magnetic media such as internal hard disks or tapes, optical media such as read-and-write compact disks, or other memory storage devices. Memory storage devices can include any of a variety of known or future devices, including compact disk drives, tape drives, removable hard disk drives, USB or flash drives, or diskette drives. These types of memory storage devices typically read from and / or write to program storage media, such as compact discs, magnetic tapes, removable hard disks, USB or flash drives, or floppy diskettes. Any of these program storage media, or others now in use or that may be developed in the future, may be considered computer program products. As will be appreciated, these program storage media typically store computer software programs and / or data. Computer software programs, also known as computer control logic, are typically stored in system memory and / or program storage devices used in conjunction with memory storage devices.In one embodiment, a computer program product comprising a computer-usable medium having stored thereon control logic (computer software program including program code). The control logic, when executed by a processor, causes the processor to perform the functions described herein. In other embodiments, some functions are implemented primarily in hardware, for example, using hardware state machines. Implementing hardware state machines to perform the functions described herein will be apparent to one skilled in the relevant art. Input / output controllers can include any of a variety of known devices that accept and process information from a user, whether human or machine, local or remote. 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. Output controllers can include controllers 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 a 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, when implemented in software, the instrument control and / or data processing application may be loaded and executed from system memory and / or memory storage devices. All or part of the instrument control and / or data processing application may reside in read-only memory or similar memory storage devices, and such devices do not require the instrument control and / or data processing application to first be loaded via an input / output controller.Those skilled in the relevant art will appreciate that the instrument control and / or data processing application, or portions thereof, may be loaded into system memory, cache memory, or both by a processor in a known manner for convenient execution. The computer may also include and store in system memory one or more library files, experimental data files, and an Internet client. For example, the experimental data may 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 synthesis (SBS) experiments or processes. Additionally, the Internet client may include applications usable to access remote services on other computers using a network, such as what is commonly referred to as a "web browser." In this example, some commonly employed web browsers include Microsoft Internet Explorer available from Microsoft Corporation, Mozilla Firefox from Mozilla Corporation, Safari from Apple Computer Corp., Google Chrome from Google Corporation, or other types of web browsers now known or hereafter developed in the art. In the same or other embodiments, the Internet client may also include, or be an element of, a specialized software application that can be used to access remote information over a network, such as a data processing application for biological applications.

[0062]

[0087] A network can include one or more of many different types of networks known to those skilled in the art. For example, a network can include a local or wide area network that can employ what is commonly referred to as the TCP / IP protocol suite for communication. A network can also include the networks that make up the worldwide system of interconnected computer networks commonly referred to as the Internet, or various intranet architectures. Those skilled in the relevant art will also recognize that some users in networked environments prefer to employ what are commonly referred to as "firewalls" (sometimes also called packet filters or boundary protection devices) to control information traffic to and from their hardware and / or software systems. For example, a firewall may include hardware or software elements or some combination thereof and is typically designed to enforce security policies put in place by users, such as network administrators.

[0063]

[0088] In one embodiment of the system 100 of FIG. 1 , the sample is mounted on a substrate holder (not shown in FIG. 1 ; examples of sample substrate holders that can be part of the system 100 include heater baths, Peltier heating and cooling baths, and trays that hold multiple samples. In some embodiments, an entire tray of samples is loaded into the system 100 by a user) and guided by the substrate transfer / substrate movement system 106 toward a baking / drying oven 112 for bonding the samples to microscope slides. In other embodiments, the baking / drying of the sample occurs outside of the system 100 and is loaded directly onto the sample substrate holder by a user of the system 100. Alternatively, as 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 perform a particular staining protocol are moved toward the sample using, for example, actuators associated with the bulk fluid applicator system 116 or the microfluidic applicator system 118. In some embodiments, reagents used in the fluid applicator system 116 or the microfluidic applicator system 118 can be moved from the reagent storage unit 120 using a reagent transfer system 122 and fluidly connected to the fluid applicator system 116 or the microfluidic applicator system 118.

[0064]

[0089] In some staining protocols, it may be desirable to perform antigen or target retrieval on the sample before further processing steps can be performed. In one embodiment, a substrate transfer / substrate movement system 106 is used to direct the sample toward an 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 above the boiling point of normal water. Optionally, a bulk antigen retrieval solution can be supplied to the antigen / target activation system 124 by a fluid / air supply module 126. In certain embodiments, the antigen / target retrieval system can be configured to be optimized for antigen retrieval (unmasking antigenic tissue prior to IHC) or target retrieval (unmasking nucleic acid sequences prior to ISH). In other embodiments, the disclosed antigen / target retrieval system 124 can include one or more dedicated antigen retrieval subsystems and one or more dedicated target activation subsystems.

[0065]

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

[0066]

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

[0067]

[0092] 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 may include a washing station or may simply be a location to which the microfluidic reagent applicators (not shown in FIG. 1) of the microfluidic applicator system 118 can be moved when not in use to ensure they do not dry out and become clogged during periods of inactivity.

[0068]

[0093] Additionally, a coverslipper module 134 may also be part of the system 100 for placing a coverslip over the sample once its processing protocol is complete.

[0069]

[0094] 2 illustrates an embodiment of the disclosed system 200 in which a sample support substrate 202 is continuously moved past processing modules 206, 208, 210, 212, 214, and 216 on a conveyor 204. Modules 206, 208, 210, 212, 214, and 216 are used to process samples; i.e., these modules may be used to perform one or more process steps required for a particular staining protocol. In one embodiment, at least one of processing modules 206, 208, 210, 212, 214, and 216 includes a microfluidic reagent applicator and at least one of 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 held stationary, with the conveyor 204 providing relative motion of the sample through the at least one processing module (continuously at a constant speed, at a variable speed, or in a stop-start manner). In other embodiments, at least one of the processing modules 206, 208, 210, 212, 214, and 216 is connected to an actuator (not shown) capable of providing 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 adjacent to the processing modules 206, 208, 210, 212, 214, and 216, and once the sample support substrate is positioned adjacent, the actuator moves the processing module relative to the substrate to dispense one or more fluids onto the sample. In certain other embodiments, at least one of the processing modules 206, 208, 210, 212, 214, and 216 includes a fluid aspirator; in certain other embodiments, at least one of the processing modules includes an air knife; and in certain other 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.Any of the processing modules 206, 208, 210, 212, 214, and 216 can be moved to a reagent storage unit 220 when not in use.

[0070]

[0095] 3 shows a schematic diagram of an embodiment of the disclosed system 300, including an array of stationary sample substrate holders 302 and multiple processing modules 304 / 306, 308, and 310. Also shown is a reagent storage module 312 and a microfluidic applicator management system 34. In this embodiment, the processing modules 304 / 306 include 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 a specific embodiment, the flexible fluid connections 316, 318, and 320 supply bulk fluid, air, and vacuum, respectively, to the bulk fluid applicator module 306. In a more specific embodiment, the fluid / air / vacuum supply module 304 includes multiple bulk fluid reservoirs that can be switched to supply different fluids to the bulk fluid applicator module 306. In another more specific embodiment, flexible fluid connections 316 and 320 supply compressed air to an air knife mounted on bulk fluid applicator module 306 and supply vacuum to a fluid aspirator mounted on 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 delivering detection chemistry reagents to samples mounted on sample substrate holders within array 302, with processing module 310 delivering the reagents, e.g., primary antibodies. In this embodiment, processing module 310 travels to reagent storage module 312 (which may be cooled and / or humidified) and retrieves a microfluidic reagent applicator containing integrated reagent reservoirs corresponding to the primary antibodies to be dispensed onto specific samples in the array according to a planned staining protocol. Once dispensing of the primary antibodies is complete, the applicator is returned to reagent storage module 312, perhaps after being sent to microfluidic applicator management system 314 for cleaning / blotting or humidification (such as by applying a moisturizer). Processing module 308 in this embodiment may include a single microfluidic reagent applicator fluidly connected to multiple integrated reagent reservoirs, as shown in FIG. 8 .

[0072]

[0097] In other embodiments, and referring again to FIG. 3, the sample substrate holder array 302 can be a 1xn, 2xn, 3xn, up to an nxn array, such as a 1x20 array, a 2x15 array, or a 3x10 array to provide 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, which can be arranged in any possible array to reach a total number of sample substrate holders. Some or all of these substrate holders can be equipped with a heater base or a Peltier heating / cooling base.

[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 can be moved across a sample support substrate 410 to treat all or a portion of a sample.

[0074]

[0099] 4B shows 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] Figure 5A shows another embodiment of a bulk fluid applicator module 500 configured for treatment of a sample disposed on a substrate. 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 fluid away from the sample (such as to the waste management system 130 of Figure 1). The embodiment of Figure 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. 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 away from the sample (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 the x, y, and z coordinate directions in addition to being moved by an actuator as shown. This embodiment of the bulk fluid applicator module is also 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, as with the bulk fluid applicator module 500 of FIG. 5A. However, the entire module can also be rotated, allowing, for example, a portion of a sample to be treated in a circular pattern. For example, the bulk fluid applicator module 520 can be used to form circular or similarly shaped wells in the paraffin of a paraffin-embedded tissue sample.

[0077]

[0102] FIG. 6A illustrates one 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. Microfluidic reagent applicator 600 can be disposable or refillable, and although shown as a collection of four individual applicators, one or more (e.g., corresponding to each step of a staining protocol, which number could be five or more, ten or more, or even twenty or more) can be combined into a single unit for use, for example, with substrate transfer / substrate movement system 204 of FIG. 2 . Alternatively, the collection of individual applicators can be used to simultaneously treat multiple samples, e.g., five or more, ten or more, or even twenty or more, of some or all of a collection of substrate-mounted samples held in a tray holding such substrate-mounted samples. The microfluidic reagent applicator according to this embodiment can be added to and removed from an automated staining system in an entirely manual process, or can be used in conjunction with a reagent transfer system for loading and unloading through access ports in an enclosure covering the staining system.

[0078]

[0103] FIG. 6B illustrates another embodiment of a 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. Microfluidic reagent applicator reservoirs 612a, 612b, 612c, and 612d can be disposable or refillable, and although shown as a collection of four individual applicators in FIG. 6B, one or more (e.g., corresponding to each step in a staining protocol; this number can be five or more, ten or more, or even twenty or more) can be combined into a single unit for use with substrate transfer / substrate movement system 204, for example, as shown in FIG. 2. Alternatively, a collection of individual applicators can be used to simultaneously treat multiple samples, e.g., five or more, ten or more, or even twenty or more, of a collection of substrate-mounted samples held in a tray holding such substrate-mounted samples. Replaceable reagent reservoirs 612a, 612b, 612c, and 612d can be added and removed from the automated staining system in a totally manual process, or can be used in conjunction with a reagent transfer system for loading and unloading through access ports in an enclosure covering the staining system.

[0079]

[0104] 6C illustrates yet another embodiment of a microfluidic reagent applicator 620 according to the present disclosure, in which remote reagent reservoirs 622a, 622b, 622c, and 622d are connected to corresponding fluid reservoirs through fluid lines 626a, 626b, 626c, and 626d. The microfluidic reagent applicator reservoirs 622a, 622b, 622c, and 622d are fluidly coupled to droplet-on-demand microfluidic actuators 624a, 624b, 624c, and 624d. Microfluidic reagent applicator reservoirs 622a, 622b, 622c, and 622d can be disposable or refillable, and although shown in FIG. 6C as a collection of four individual applicators, one or more (e.g., corresponding to each step of a staining protocol; this number could be 5 or more, 10 or more, or 20 or more) can be combined into a single unit for use, for example, with the substrate transfer / substrate movement system 204 of FIG. 2. Alternatively, the collection of individual applicators can be used to simultaneously treat multiple samples, e.g., some or all of a collection of substrate-mounted samples held in a tray holding such substrate-mounted samples, such as 5 or more, 10 or more, or 20 or more. Fluid lines 626a, 626b, 626c, and 626d can be rigid, flexible, or a combination of both rigid and flexible, both between lines and within a single line. The embodiment of Figure 6C, when the lines are flexible, is also suitable for simultaneously processing all or part of an array of samples, as shown in Figure 3. Such a configuration also provides the opportunity to refill the reservoirs from a location external to the device, and therefore may be particularly suitable for bulk fluid dispensing.

[0080]

[0105] 6D illustrates yet another embodiment of a 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 lines 636a, 636b, 636c, and 636d. This embodiment also includes intermediate reservoirs 638a, 638b, 638c, and 638d. Microfluidic reagent applicator reservoirs 632a, 632b, 632c, and 632d can be disposable or refillable, and although shown in FIG. 6D as a collection of four individual applicators, one or more (e.g., corresponding to each step in a staining protocol, and this number can be 5 or more, 10 or more, or 20 or more) can be combined into a single unit for use, for example, with substrate transfer / substrate movement system 204 of FIG. 2. Alternatively, a collection of individual applicators can be used to simultaneously treat multiple samples, e.g., 5 or more, 10 or more, or 20 or more, of some or all of a collection of substrate-mounted samples held in a tray holding such substrate-mounted samples. Fluid lines 636a, 636b, 636c, and 636d can be rigid, flexible, or a combination of both rigid and flexible, both between lines and within a single line. The embodiment of Figure 6C, if the tubing is flexible, is also suitable for simultaneously processing all or part of an array of samples, as shown in Figure 3. Such a configuration also provides the opportunity to refill the reservoirs from a location external to the instrument, and therefore may be particularly suitable for bulk fluid dispensing. Furthermore, intermediate reservoirs 638a, 638b, 638c, and 638d allow for easy on-the-fly replacement of remote reagent reservoirs 632a, 632b, 632c, and 632d without interrupting sample processing within the automated staining system.

[0081]

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

[0082]

[0107] FIG. 7 schematically illustrates a reagent transfer system 700 that can be used to transfer a replaceable fluid reservoir 704 (as shown in and described with reference to FIG. 6B ) to and from a reagent storage unit 720 once it has been disconnected from the microfluidic actuator 702. The reagent storage unit 720 can be cooled to extend reagent life while not in use. It also illustrates how a microfluidic reagent applicator (as shown in and described with reference to FIG. 6A ) including an integrated reservoir 706 can be shuttled to and from the reagent storage unit 720 by the reagent transfer system 700. Furthermore, FIG. 7 illustrates how a remote reagent reservoir 708 can be held in storage and used to supply a microfluidic reagent applicator 710, as needed, through tubing 712 and optional intermediate reservoirs (as shown in and described with reference to FIGS. 6C and 6D ).

[0083]

[0108] FIG. 8 illustrates a microfluidic reagent applicator system 800 including a droplet-on-demand actuator head 802 fluidly connected to multiple reagent reservoirs 804a, 804b, 804c, and 804d. Also illustrated in FIG. 8 is an embodiment of a sample substrate holder 806 in which a substrate 808 supporting a sample 810 rests on a base 812. The base can be a heater base or a Peltier heating / cooling base. Where compatible, reagents contained in multiple reagent reservoirs 804a, 804b, 804c, and 804d can be sequentially dispensed to perform a staining protocol on the sample 810. This simple configuration is suitable for point-of-care systems, such as those used in operating rooms to stain frozen tissue samples. Such a point-of-care system can be used, for example, by a surgeon to determine whether he / she successfully removed a tumor from a patient beyond its resection margins. Reagent reservoirs 804a, 804b, 804c, and 804d can be integral with droplet-on-demand actuator head 802, can be interchangeable with droplet-on-demand actuator head 802, or can be remotely located and fluidly connected via tubing.

[0084]

[0109] FIG. 9A shows a front view of an embodiment of an integrated system including 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 a 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 is 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 aspiration port 910, which is also shown in the side view of FIG. 9B. FIG. 9C is a bottom view of the 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 a 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, including 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 combined bulk fluid applicator and fluid aspirator 1004 is mechanically coupled to the microfluidic reagent applicator head 1006. As shown in the front view of FIG. 10A, the integrated system includes a bulk fluid inlet 1008 and a fluid aspiration port 1010, which is also shown in the side view of FIG. 10B. As shown in the front view of FIG. 10A, the integrated system includes a bulk fluid applicator needle 1012 and a fluid aspiration needle 1014, which is also shown in the side view of FIG. 10B. 10C shows a bottom view of this integrated system, illustrating the dual-needle combined bulk fluid applicator and fluid aspirator 1004, including a bulk fluid applicator needle 1012 and a fluid aspirator needle 1014. Also shown in FIG. 10C is the microfluidic droplet-on-demand array 1016 of the microfluidic reagent applicator head 906.

[0086]

[0111] FIG. 11 illustrates one staining subsystem 1000 for use alone or in combination with additional such same and other subsystems according to the present disclosure. In this embodiment, a microfluidic reagent applicator 1102 is mechanically coupled to an actuator 1104. In certain embodiments, a substrate holder 1108 is also included, which holds a sample support substrate 1110. The substrate holder 1108 is also mechanically coupled to an actuator 1112. In certain embodiments, a combined bulk fluid applicator and air knife module 1106 is also included. One or more waste collection units 1114 (which may 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 the actuator 1104 then brings the microfluidic reagent applicator 1102 into position to dispense fluid onto the sample. Depending on which reagents are to be dispensed onto the sample, actuator 1112 moves the substrate holder and substrate to one of at least two different positions 1108b, 1110b, or 1110c so that a combined bulk fluid applicator and air knife (such as at positions 1106a, 1106b, or 1106c, or at any intermediate position not shown) can, for example, apply a rinse solution and then displace or "blow" this rinse solution and any reagent residue away from the sample into a respective waste collection unit 1114a, 1114b. It will be appreciated by those skilled in the art that, while in some embodiments the bulk fluid applicator and air knife move with the substrate holder, in other embodiments the bulk fluid applicator and air knife are separate units that are not connected to (and can move in and out of) the substrate holder. In an alternative embodiment, an entire row of microfluidic reagent applicators can be translated back along the axis of rotation of actuator 1104 so that additional reagent can be brought into dispensing / loading position relative to the sample.Although the dispensing position is shown horizontal in Figure 11, alternative configurations are possible in which the sample support substrate is held in a position other than horizontal, such as between about 1 degree and about 90 degrees from horizontal, or in which it is held upside down during dispensing of fluid from the microfluidic reagent applicator.

[0087]

[0112] Figure 12 shows a top view of the embodiment of Figure 11, where the combined bulk fluid applicator / air knife module is shown as 1206. A substrate holder 1208 holds a sample support substrate 1210, and module 1206 moves across the sample support substrate 1210 by a screw drive 1224 powered by a motor 1228. Module 1206 is also guided by rails 1226 and held in place by posts 1230. Air is supplied to module 1206 through port 1220, and bulk fluid is supplied to module 1206 through port 1222.

[0088]

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

[0089]

[0114] FIG. 14 illustrates a specific embodiment of a substrate holder system 1400 that can be used in conjunction with a microfluidic reagent dispenser to form a chamber above a sample held on a substrate. Here, a microfluidic reagent actuator 1404 mates with a substrate holder 1406 and further mates with seals 1412a and 1412b. Shown inside are a substrate holder 1408 and a sample support substrate 1410. In one embodiment, the microfluidic reagent actuator is a thermal droplet-on-demand system. In a specific embodiment, a fluid reservoir 1402 contains an antigen retrieval solution, which is dispensed onto the sample within the chamber. In some embodiments, and provided a vent is included, the contents dispensed onto the sample may be heated and then replenished with additional fluid. In other embodiments, the chamber may be pre-pressurized, or pressure may build up within the chamber as heat is applied.

[0090]

[0115] Applicant has surprisingly discovered that the systems and methods disclosed herein can be used to co-dispense hematoxylin and eosin, which are generally incompatible in solution. Compared to linear assays, co-dispensing using the systems and methods disclosed herein allows for a significant reduction in the number of assay steps (e.g., from 10 to 5 steps) and a concomitant reduction in total assay volume (e.g., from about 2.44 mL / slide to about 1.26 mL / slide) while achieving similar results.

[0091]

[0116] Figures 15A and 15B show two embodiments of deparaffinization according to the present disclosure, both of which compare favorably with standard methods in terms of incubation time and volume. The figures show printed deparaffinization: printing of organic matter and transition fluid onto paraffin sections, followed by bulk washing with the solutions. Co-printing or sequential modes were found to be possible. Assays required only small volumes (approximately 100 uL / in²) and little or no incubation time (approximately 2 minutes to print). A single microfluidic deparaffinization process using a dual-needle and dual-slit (as described herein) could also be utilized, again requiring only small volumes and little or no incubation time (approximately 2 minutes to print).

[0092]

[0117] While particular configurations of bulk fluid applicators, fluid applicators, and air knives have been disclosed, other configurations are possible. For example, the system described in U.S. Pat. No. 8,883,509 can be used in combination with other features disclosed herein. Similarly, the particular bulk fluid handling and reagent dispensing mechanism disclosed in WO 2015 / 086534 can be used in combination with other features disclosed herein.

[0093]

[0118] Additional Embodiments

[0094]

[0119] The various components shown in Figure 16 are described below. In one embodiment, the control system or method includes a "print command and map generator," which functions to generate combinatorial two- (or three-) axis motions in conjunction with nozzle firing for directed delivery of droplets to obtain the desired image on the droplet target. Generally, this is a mapping between a binary (or monochrome) bitmap file, where one state (i.e., logical true) for each pixel in the file corresponds to one droplet 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 command for the motion system, which in turn is the command for the print system.

[0095]

[0120] In one embodiment, the control system or strategy includes a "Digital Firing Pulse Generator" whose function is to resolve the print map in the context of both the relative motion system and the assignment of individual droplet dispensing actions to corresponding nozzles.

[0096]

[0121] In one embodiment, the control system or strategy includes a "relative motion system" that facilitates motion of the target relative to the print head according to a set of commands (i.e., print instructions) sent from the print instruction and map generator. Information about the system's motion relative to a reference position is relayed to a digital fire pulse generator via a step counter to monitor progress in mapping the image onto the physical print, and correspondingly, when to deliver droplets for deposition on the droplet target during that print.

[0097]

[0122] In one embodiment, the control system or method includes a "step counter" that provides the digital firing pulse generator with an understanding of the motion and relative position of the target and print head to train the deposition of droplets by the relative motion system.

[0098]

[0123] In one embodiment, the control system or method includes a "waveform generator" that initiates a droplet dispense act when a logical true is issued for a particular nozzle address. This function converts that signal into the analog signals needed to prime, dispense, and refill the nozzle for successful droplet ejection.

[0099]

[0124] In some embodiments, the control system or strategy includes an "amplifier": the generated waveform is amplified to an appropriate range of excitation potentials (i.e., voltages or energies) to induce droplet ejection.

[0100]

[0125] In some embodiments, the control system or method includes a "fluid droplet dispenser." The amplified signal is passed to a corresponding nozzle, which fires a droplet onto a droplet target in step with a relative motion system.

[0101]

[0126] In one embodiment, the control system or method includes a "droplet target": a final target that receives droplets from a fluid droplet dispenser and is moved in unison by a relative motion system to map the print map onto the target.

[0102]

[0127] The various physical control methods are outlined in Figure 17. In one embodiment, the control system or method includes a CPU. In this case, the CPU is a desktop computer. The CPU's role is to coordinate the issuance of commands to the physical system through print instructions and print maps. These are derived from monochrome bitmap files, resulting in the desired drop print density in the x and y axes. The x-axis print density is physically determined by setting the encoder step size on the Print Manager Board, and the y-axis print density is set by adjusting the saber angle of the print head. For example, a 1000 x 1000 pixel image printed at 1000 x 1000 dpi will produce a 1 in2 print. Similarly, printing the same image at 500 x 500 dpi will produce a 2 in2 print. Finally, printing the same image at 2000 x 2000 dpi will produce a 0.5 in2 print. As part of the print command, the CPU provides velocity information to the relative motion system, as well as the y-step and start position for the print job, fixing the printing system in a "raster print" configuration, which translates the image into print on the print target by a continuous print move in the x direction followed by an incremental y-step move prior to the next print pass in the x direction.

[0103]

[0128] In one embodiment, the control system or control strategy contains information about the print job to coordinate the print instructions, the relative motion system, and the print manager board.

[0104]

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

[0105]

[0130] In one embodiment, the control system or scheme includes a print manager board, which performs the role of the digital fire pulse generator function in the previous chapter. The board parses the print map and assigns nozzles to fire for specific pixels in the map. Given information about the encoder step size, it determines how long to wait for an encoder pulse between each pixel in the print map. Generally, there is a one-to-many relationship between this board and the driver cards, with the driver cards effectively acting as daughter boards to the higher-level functions running on the print manager board.

[0106]

[0131] In some embodiments, the control system or strategy includes a relative motion system, as described above in the previous section.

[0107]

[0132] In one embodiment, the control system or method includes a digital fire signal: when the print manager board determines that a drop dispensing operation should occur (by resolving the print map and monitoring the print head position using the encoder step pulses as feedback), it issues a command to a nozzle (or multiple nozzles simultaneously) by issuing a fire signal down a corresponding data path to the nozzle driver card.

[0108]

[0133] In one embodiment, the control system or scheme includes driver cards and performs the waveform generator and amplifier functions described in the previous chapters. It is responsible for power management, eliminating crosstalk between the electrical paths of firing nozzles, and returning print nozzles to a ready state for re-firing. Typically, there is a one-to-one mapping with the print head, and the driver cards provide dedicated signal lines for each nozzle on the print head. However, the actual architecture can also support a one-to-many association with print heads, since each signal line is mapped to a nozzle, regardless of whether those nozzles are co-located on the same print head.

[0109]

[0134] In some embodiments, the control system or method includes vector graphics. In other embodiments, a plotter approach and a vector graphics (e.g., PostScript) language for describing print operations can offer certain advantages. Vector graphics describe lines and arcs for printing instead of pixels. This allows for infinite scaling and translation of the print image without loss of resolution. It also allows for complex coordinated movement of the print system. Print operations can be programmatically defined (functions, loops, variables, etc.) rather than statically defined by an image file. In this environment, for example, print vectors can be defined for specific lengths, speeds, firing frequencies, and print swath widths.

[0110]

[0135] Advantages (as shown in Figure 17) refer to a common framework for describing all motion components for any operation the stainer must perform (washing, drying, etc.); accommodate complex 2- or 3-axis printing operations; no need to perform sequential printing operations in the x-direction followed by a step in the y-direction; ease of extending and transforming the tissue image into the print area, and ease of reusing images from one slide to print other stains on other slides with the same tissue cut.

[0111]

[0136] Multicolor bitmaps. Instead of individual single-color bitmaps being applied to the sample per print head and per printed image, 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 can be 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] Advantages include a precise method for defining the simultaneous use of multiple print heads or for describing the temporal redistribution of reagents by printing (as shown in Figure 17). The use of common image storage languages ​​such as JPEG, TIFF, JPEG-2000, etc. allows for compression as opposed to bitmap file formats.

[0113]

[0138] Various other modifications of the disclosed systems and methods, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description, and such modifications are intended to fall within the scope of the appended claims. For example, while the above disclosure has focused on the processing 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 herein is incorporated by reference in its entirety to the extent that it does not contradict this disclosure.

Claims

1. 1. An automated biological specimen staining system, comprising: a. at least one microfluidic reagent applicator; b. at least one bulk fluid applicator; c. at least one fluid aspirator; d. at least one sample substrate holder; e. at least one relative motion system; f. a control system programmed to execute at least one staining protocol on a sample mounted on a substrate held in said at least one sample substrate holder; wherein the control system controls the at least one microfluidic reagent applicator, the at least one bulk fluid applicator, the at least one fluid aspirator, the at least one sample substrate holder, and the at least one relative motion system to perform individual steps of the at least one staining protocol.

2. 10. The system of claim 1, further comprising at least one sample imaging system.

3. 10. The system of claim 1, further comprising at least one air knife.

4. 10. The system of claim 1, further comprising at least one waste management system.

5. 10. The system of claim 1, further comprising at least one sample identification system communicatively coupled to the control system.

6. 10. The system of claim 1, wherein the at least one bulk fluid applicator and the at least one fluid aspirator are combined with at least one first type reagent management unit.

7. The system of claim 6 , wherein the at least one bulk fluid applicator and the at least one fluid aspirator comprise a pair of needles.

8. 8. The system of claim 7, wherein the pair of needles are separated by at least 0.1 mm.

9. 10. The system of claim 1, wherein the at least one microfluidic reagent applicator, the at least one bulk fluid applicator, and the at least one fluid aspirator are combined with at least one second type reagent management unit.

10. 3. The system of claim 2, wherein 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 reagent management unit.

11. 3. The system of claim 2, wherein the at least one microfluidic reagent applicator, the at least one bulk fluid applicator, the at least one fluid aspirator, and the at least one air knife are combined with at least one fourth type reagent management unit.

12. 12. A system according to any one of claims 6, 9, 10 and 11, wherein at least one of any of the first, second, third and fourth types of reagent management units is coupled to the at least one relative motion system.

13. 12. The system of any one of claims 6, 9, 10 and 11, wherein said at least one sample substrate holder is coupled to said at least one relative motion system.

14. 12. A system according to any one of claims 6, 9, 10 and 11, wherein at least one of any of the first, second, third and fourth types of reagent processing units and the at least one sample substrate holder are both coupled to the at least one relative motion system.

15. The system of claim 1 , wherein the system comprises two or more microfluidic reagent applicators.

16. 10. The system of claim 1, further comprising a refrigerated reagent storage unit.

17. The system of claim 1 further comprising a reagent transfer system.

18. The system of claim 1 , wherein the system includes two or more bulk fluid applicators and two or more fluid aspirators.

19. 10. The system of claim 1, wherein the at least one microfluidic reagent applicator comprises a microfabricated tip applicator.

20. 10. The system of claim 1, wherein the at least one microfluidic reagent applicator comprises a droplet-on-demand actuator.

21. 21. The system of claim 20, wherein the droplet-on-demand actuator comprises a piezoelectric actuator.

22. 21. The system of claim 20, wherein the droplet-on-demand actuator comprises a thermal actuator.

23. 21. The system of claim 20, further comprising at least one piezoelectric actuator and at least one thermal actuator.

24. 10. The system of claim 1, wherein the microfluidic reagent applicator comprises an integrated reagent reservoir in fluid communication with a droplet-on-demand actuator.

25. 10. The system of claim 1, wherein the microfluidic reagent applicator comprises a remote reagent reservoir in fluid communication with a droplet-on-demand actuator.

26. 10. The system of claim 1, wherein the microfluidic reagent applicator comprises a droplet-on-demand actuator and a replaceable reagent reservoir, and when the droplet-on-demand actuator and the replaceable reagent reservoir are coupled, the replaceable reagent reservoir is fluidly connected to the droplet-on-demand actuator.

27. 10. The system of claim 1, wherein the microfluidic reagent applicator comprises a droplet-on-demand actuator integrated with an intermediate reagent reservoir, and the microfluidic reagent applicator unit is in fluid communication with a remote reagent reservoir.

28. 1. A method for automated handling of at least a portion of a sample held on a substrate, said method comprising: a. obtaining an image of the sample on the substrate; b. automatically locating the sample on the substrate; c. dispensing a fluid at the location of the sample on the substrate; A method comprising:

29. 30. The method of claim 28, wherein dispensing a fluid at the location of the specimen on the substrate comprises dispensing the fluid substantially only at the location of the specimen on the substrate.

30. 30. The method of claim 28, further comprising removing the fluid from the site on the substrate where the sample is located.

31. 30. The method of claim 28, wherein the sample comprises a paraffin-embedded sample, and automatically locating the sample on the substrate comprises automatically detecting a portion of the paraffin section containing the sample.

32. 32. The method of claim 31, wherein the fluid comprises a deparaffinizing fluid, and wherein the step of pouring the fluid comprises pouring the deparaffinizing fluid into at least one small portion of the paraffin section that contains a small portion of the sample.

33. 33. The method of claim 32, further comprising the step of removing the deparaffinizing fluid from at least one small portion of the paraffin section containing a small portion of the sample, to form a well in the paraffin surrounding the small portion of the sample.

34. 32. The method of claim 31, wherein the fluid comprises a deparaffinizing fluid, and wherein the step of pouring the fluid comprises pouring the deparaffinizing fluid substantially onto the portion of the paraffin section containing the sample.

35. 35. The method of claim 34, further comprising the step of substantially removing the deparaffinizing fluid from the portion of the paraffin section containing the sample, leaving paraffin on the substrate around the sample to form a well in the paraffin substantially surrounding the sample.

36. 36. The method of any one of claims 33 or 35, wherein the steps of pouring out the deparaffinization fluid and removing the deparaffinization fluid are performed simultaneously.

37. 36. The method of claim 33 or 35, further comprising dispensing at least one second fluid into the well in the paraffin surrounding the small portion of the sample or into the well in the paraffin substantially surrounding the sample, respectively.

38. 38. The method of claim 37, wherein dispensing at least one second fluid into the wells in the paraffin comprises dispensing one or more of water, a buffer solution, an antibody solution, a dye solution, a nucleic acid solution, a solvent, a surfactant, and a humectant into the wells in the paraffin.

39. 29. The method of claim 28, further comprising the steps of selecting two or more distinct sub-portions of the sample from the location of the sample on the substrate, and dispensing the deparaffinizing fluid onto the two or more distinct selected sub-portions of the sample to dissolve paraffin on the two or more distinct selected sub-portions of the sample.

40. 40. The method of claim 39, further comprising removing the deparaffinizing fluid from the two or more separate sub-portions of the sample to form two or more separate wells in the paraffin surrounding the two or more separate sub-portions of the sample.

41. 40. The method of claim 39, wherein selecting two or more distinct sub-portions of the sample comprises selecting two or more distinct sub-portions from images of H&E stained serial sections of the same sample block from which the sample was obtained to reveal morphological features of the serial sections; and mapping the morphological features from the serial sections to the sample on the substrate.

42. 42. The method of claim 41, wherein the two or more distinct sub-portions of the sample mapped from the serial sections are selected to correspond to different morphological features of the sample.

43. 42. The method of claim 41, wherein the two or more distinct sub-portions of the sample mapped from the serial sections are selected to provide at least one of a positive control and a negative control, and further to provide at least one sub-portion of the sample for comparison with the at least one of the positive control and the negative control.

44. 37. The method of claim 36, wherein the step of simultaneously injecting the deparaffinization fluid and removing the deparaffinization fluid includes the step of injecting the deparaffinization fluid from a first needle, and the step of removing the deparaffinization fluid includes the step of aspirating the deparaffinization fluid into a second needle positioned at least 0.1 mm from the first needle.

45. 45. The method of claim 44, wherein the first and second needles translate linearly together across the sample.

46. 45. The method of claim 44, wherein the first and second needles rotate about a common axis.

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