Apparatus and method for processing biological samples

Abstract

A method and apparatus for processing samples on slides is provided. A capillary processing module has a crank that raises and lowers the slide, and / or a side inlet that applies fluid to the slide. When the crank lowers the slide, a capillary gap is formed between the slide and the chamber floor of the capillary processing module. Capillary action causes fluid to spread through the capillary space across the processing area and contact the sample. When the crank raises the slide, the capillary gap disappears, allowing fluid to be drawn from the processing area of ​​the slide.

Description

[Technical Field]

[0001] The present invention relates to an apparatus and method for processing biological samples on slides using a capillary processing module.

[0002] [CROSS-REFERENCE TO RELATED APPLICATIONS] Not applicable [Background technology]

[0003] Sample processing for histology, cytology, molecular pathology, biochemistry, immunology, microbiology, and other chemical and biological analyses can involve one or more processing steps on the sample. The sample can be placed on a slide before performing some or all of the desired processing steps. For example, the sample on the slide can be contacted with one or more reagents that label and / or react with components of the sample (i.e., label and / or react with components of the sample). A wide variety of processing steps are known in these fields and / or can be developed to perform a particular analysis (i.e., a wide variety of processing steps are known, or the processing steps can be developed to perform a particular analysis, or both).

[0004] Many reagents used in processing biological samples are expensive, difficult to obtain, and / or potentially hazardous (i.e., expensive, difficult to obtain, and / or potentially hazardous), and therefore it is desirable to reduce or minimize the amount of reagent used in processing the sample. In the case of liquid reagents, it is often desirable to reduce the volume of reagent utilized in processing the sample while maintaining the concentration of the reagent dissolved in the liquid. In the case of biological samples on slides, it is difficult to use smaller volumes of liquid reagents because the smaller volumes may not be able to completely cover the sample, resulting in inconsistent or improper processing of the sample.

[0005] One way to increase sample coverage by a liquid reagent is to spread the reagent over an area of ​​the slide by forming a capillary gap between the flat surface and the opposing surface of the slide. The liquid in such a capillary gap tends to spread into and fill the capillary space due to capillary forces, thereby covering the flat surface of the slide and the sample thereon. However, once the liquid spreads and fills the capillary space, further movement of the liquid in the capillary gap is restricted by capillary forces. Passive mixing and redistribution of the liquid in the capillary gap can be delayed or inadequate. When the reagent dissolved in the liquid is consumed by reacting with or binding to the sample placed on the slide, a region of low reagent concentration may form around the sample. Unless the liquid reagent in this region is replenished by mixing, redistributing, or replacing the liquid reagent in this space with new reagent, the consumption of the reagent by the sample delays and extends the time required to complete the process. Furthermore, density differences between low density regions and other regions can result in inconsistent processing across a range of samples, producing undesirable effects such as staining gradients.

[0006] Some drawbacks associated with conventional slide processing devices are that they require relatively large volumes of processing liquid, their performance is inferior to manual processing, and / or they are relatively complex or require a large number of moving parts (i.e., they require relatively large volumes of processing liquid, or their performance is inferior to manual processing, or they are relatively complex or require a large number of moving parts, or all of these). Other drawbacks are that these devices are unable to rapidly heat or cool the slides and / or they create temperature gradients around the slides (i.e., they are unable to rapidly heat or cool the slides, or they create temperature gradients around the slides, or both).

[0007] Patent Document 1 to Larsen et al. describes a method and automated device for processing at least one biological sample placed on a slide. At least one capillary staining module includes a slide rack holder configured to removably hold a slide rack configured to hold slides, and a capillary lid rack holder configured to removably hold a capillary lid rack configured to hold capillary lids, and the slide rack can be removed independently of the capillary lid rack. The first fluid chamber contains a first fluid. The device is configured to automatically rotate one or more slides and move the lid toward the slides to automatically form capillary gaps between each slide and each capillary lid, functioning as capillary chambers, and supply a quantity of the first fluid to the slide. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] U.S. Patent No. 10,018,542 Summary of the Invention

[0009] In one aspect, the present disclosure provides a method for processing a biological sample on a slide. The method includes placing the slide in a capillary processing module. The capillary processing module includes a chamber having a chamber floor and a crank positioned to raise and lower a portion of the slide relative to the chamber floor. The method also includes rotating the crank to raise and lower the portion of the slide. When the slide is in the lowered position, a capillary gap is formed between the chamber floor and the slide. By way of example, the crank can raise and lower a first end of the slide while a second end of the slide remains on the chamber floor or on a slide support extending from the chamber floor.

[0010] In another aspect, the present disclosure provides a capillary processing module (CPM). The CPM includes a chamber having a chamber floor and one or more chamber sidewalls (e.g., one continuous chamber sidewall or four chamber sidewalls joined by corners). The CPM also includes a crank positioned to raise and lower a portion of a slide when rotated. The crank can have a curved portion and / or an asymmetrical portion (i.e., a curved portion or an asymmetrical portion, or both) positioned to engage and disengage with the slide when the crank is rotated. A capillary gap is formed between the chamber floor and the slide when the slide is in the lowered position.

[0011] The capillary treatment module may also include a chamber lid for the chamber. The chamber lid is movable between an open position and a closed position (such as by a chamber hinge connecting the lid and the chamber). The CPM may also include a port on the chamber lid, a port plug configured to seal the port, and a plug arm having the port plug on an end thereof, the plug arm configured to move the port plug toward, away from, or into the port. The CPM may also include one or more heaters on the chamber and / or on the chamber lid (i.e., on the chamber or the chamber lid, or both), and / or one or more cooling devices (e.g., blowers) positioned outside the chamber and below the chamber floor (i.e., one or more heaters on the chamber and / or on the chamber lid, and / or one or more cooling devices positioned outside the chamber and below the chamber floor). The CPM may also include one or more temperature sensors on the chamber and / or on the chamber lid. The capillary treatment module may include an outlet in the chamber and a pump fluidly connected to the outlet. The exhaust port can be located at an end of the chamber opposite the crank. The chamber of the CPM can have a first recess and a second recess separated by a processing region, the first recess having the crank shaft disposed therein and the second recess having the exhaust port disposed therein.

[0012] In some embodiments of the method and apparatus, the first end of the slide is elevated or raised so that the surface of the slide and the chamber floor form an angle between 0 and 15 degrees, or between 0 and 11 degrees, or between 0 and 2 degrees. A capillary gap is formed between the chamber floor and the slide by rotating the crank to lower the slide. A volume of a first fluid can be applied to the chamber floor when the slide is in the lowered or raised position, preferably by applying fluid at an inlet of the capillary staining module. The first fluid spreads across the processing area of ​​the slide by capillary force when the slide is in the lowered position or moving toward the lowered position. The slide can be elevated by rotating the crank, and the first fluid is drawn out of the processing area by the elevation of the slide. The method can include repeatedly raising and lowering the slide by rotating the crank unidirectionally and / or bidirectionally (i.e., unidirectionally, bidirectionally, or both), thereby drawing out and spreading the first fluid, such as to mix the fluids or prevent localized depletion of a reagent. In some embodiments of the present methods and apparatus, the first fluid is removed from the CPM through the outlet by rotating a crank to raise the slide and activating a mechanism (such as a pump) that provides suction at the outlet. In some embodiments, the first fluid is removed by activating the mechanism while the slide is in the lowered position, removing substantially all of the fluid from the chamber (except for the volume formed between the slide support and the chamber floor), after which the slide is raised by rotating a crank and suction continues through the outlet to remove any remaining fluid.

[0013] These and other features and advantages of the present methods and apparatus will become apparent from the following detailed description, taken in conjunction with the appended claims.

[0014] The present teachings are best understood from the following detailed description when read in conjunction with the accompanying drawings, the features of which are not necessarily drawn to scale. [Brief explanation of the drawings]

[0015] [Figure 1A] FIG. 1 is a conceptual diagram of one embodiment of a crank for use in the present apparatus and method. [Figure 1B] FIG. 1 is a conceptual diagram of one embodiment of a crank for use in the present apparatus and method. [Figure 1C] FIG. 1 is a conceptual diagram of one embodiment of a crank for use in the present apparatus and method. [Figure 1D] FIG. 1 is a conceptual diagram of one embodiment of a crank for use in the present apparatus and method. [Figure 2A] FIG. 10 is a diagram of another embodiment of the present apparatus and method. [Figure 2B] FIG. 10 is a diagram of another embodiment of the present apparatus and method. [Figure 2C] FIG. 10 is a diagram of another embodiment of the present apparatus and method. [Figure 2D] FIG. 10 is a diagram of another embodiment of the present apparatus and method. [Figure 3] FIG. 1 is a perspective view of an embodiment of a CPM according to the present disclosure. [Figure 4] 1A-1C are diagrams of several different crank designs for use in the present apparatus and methods. [Figure 5A] 10A-10C are diagrams of another embodiment of the present apparatus and method in which the crank has an asymmetric portion. [Figure 5B] 10A-10C are diagrams of another embodiment of the present apparatus and method in which the crank has an asymmetric portion. [Figure 5C] 10A-10C are diagrams of another embodiment of the present apparatus and method in which the crank has an asymmetric portion. [Figure 6A] 10A-10C are diagrams of various elements of another embodiment of the present apparatus and method, particularly the chamber of a CPM. [Figure 6B] 10A-10C are diagrams of various elements of another embodiment of the present apparatus and method, particularly the chamber of a CPM. [Figure 6C] 10A-10C are diagrams of various elements of another embodiment of the present apparatus and method, particularly the chamber of a CPM. [Figure 6D]10A-10C are diagrams of various elements of another embodiment of the present apparatus and method, particularly the chamber of a CPM. [Figure 6E] 10A-10C are diagrams of various elements of another embodiment of the present apparatus and method, particularly the chamber of a CPM. [Figure 6F] 10A-10C are diagrams of various elements of another embodiment of the present apparatus and method, particularly the chamber of a CPM. [Figure 7A] 10A-10C are diagrams of another embodiment of the present apparatus and method in which the crank has an asymmetric portion. [Figure 7B] 10A-10C are diagrams of another embodiment of the present apparatus and method in which the crank has an asymmetric portion. [Figure 7C] 10A-10C are diagrams of another embodiment of the present apparatus and method in which the crank has an asymmetric portion. [Figure 8A] FIG. 1 illustrates an embodiment of a CPM with a movable plug and port. [Figure 8B] FIG. 1 illustrates an embodiment of a CPM with a movable plug and port. [Figure 8C] FIG. 1 illustrates an embodiment of a CPM with a movable plug and port. [Figure 9A] FIG. 1 illustrates an embodiment of a CPM with a chamber lid attached to the frame to facilitate automatic opening and closing of the chamber. [Figure 9B] FIG. 1 illustrates an embodiment of a CPM with a chamber lid attached to the frame to facilitate automatic opening and closing of the chamber. [Figure 9C] FIG. 1 illustrates an embodiment of a CPM with a chamber lid attached to the frame to facilitate automatic opening and closing of the chamber. [Figure 10] FIG. 10 illustrates one embodiment of a capillary staining module with a cooling mechanism. [Figure 11] FIG. 10 is a diagram of one embodiment of a temperature sensor for a capillary treatment module. [Figure 12] 10A-10C are diagrams of advantageous heater designs for the top surface of the capillary treatment module. [Figure 13] 10A-10C are diagrams of advantageous heater designs for the bottom of the capillary treatment module. [Figure 14]1 is a schematic perspective view of one embodiment of an automated staining apparatus comprising multiple capillary treatment modules. FIG. DETAILED DESCRIPTION OF THE INVENTION

[0016] Before describing various embodiments, it should be understood that the teachings of the present disclosure are not limited to the specific embodiments described. Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which this disclosure belongs. All patents and publications referenced herein are expressly incorporated herein by reference.

[0017] As used in this specification and the appended claims, the terms "approximately" and "about" have their ordinary meanings and also mean within acceptable limits or amounts to those of ordinary skill in the art. The term "about" generally refers to ±15% of the indicated number. For example, "about 10" can indicate a range of 8.5 to 11.5. For example, "approximately the same" means that the compared items would be considered the same by those of ordinary skill in the art. In this disclosure, numerical ranges are inclusive of the numbers defining the range. Each subrange between a stated value or value within a stated range and any other stated value or value within a stated range is also intended to be disclosed. When a stated range includes limits, ranges excluding either or both of those included limits are also included in the disclosure. Any range disclosed herein includes all subranges between their endpoints. As used herein, the terms "a," "an," and "the" include both singular and plural referents unless the context clearly dictates otherwise. Thus, for example, "a fluid" includes one fluid and multiple fluids. Unless otherwise indicated, the terms "first," "second," "third," and other ordinal numbers are used herein to distinguish between different elements of the devices and methods and are not intended to constrain numerical limitations. References to a first layer and a second layer should not be construed to mean that the device has only two layers. A device having a first element and a second element can also include a third element, a fourth element, a fifth element, and so on, unless otherwise indicated.

[0018] In general, it is understood that the figures and the various elements shown therein are not drawn to scale. Additionally, relative terms such as "top," "bottom," "top," "bottom," "upper," "lower," "left," "right," "vertical," and "horizontal" are used to describe the relationship of various elements to one another as may be shown in the accompanying figures. It is understood that these relative terms are intended to encompass different orientations of the device and / or elements (i.e., the device and / or elements) in addition to the orientation shown in the figures. For example, if a capillary processing module is inverted relative to the perspective of the figures, an element described as, e.g., "above" another element would now be "below" that element. Similarly, if the device is rotated 90 degrees relative to the perspective of the figures, an element described as, e.g., "vertical" would now be "horizontal."

[0019] The term "crank" refers to any mechanism or device that converts rotary motion into linear motion and / or vice versa (i.e., rotary motion into linear motion and / or vice versa). A crank generally has a shaft configured to rotate and a portion that moves through a larger arc than another portion of the shaft. In some embodiments, the bent portion is bent at a right angle, as shown in FIG. 4. The bent portion may be open or form a closed loop. A crank can include an arm that is rotated or engages an actuator for rotation. The arm may be integral with the shaft or a separate piece attached to the shaft. A crank can rotate directly or indirectly, such as when the arm physically contacts the shaft and a rotational force is applied, or when a magnetic force rotates the shaft. Rotation of a crank can include a complete rotation or a partial rotation, such as over a limited arc, including moving the shaft back and forth less than a complete rotation, such as by pivoting through an arc of about 90 degrees. In some embodiments, the shaft has a portion with a circular, elliptical, oval, or non-circular cross-sectional shape. Various shapes, such as oblong, polygonal, square, rectangular, etc., may be selected as appropriate for use.

[0020] In some embodiments, the crank comprises arms connected to a shaft at an angle, such as a right angle, to facilitate rotation. The shaft can have a bent or asymmetrical section, such as an axially offset integral bend in the middle of a U-shape. The shaft can also have axially aligned ends.

[0021] The crank typically includes a bearing, and the chamber typically includes a bearing hole on one or two chamber side walls on either side of the CPM. The bearing holes can be through holes or blind holes, and the CPM may have one of each. The crank can include one or more journals, i.e., portions of the shaft specifically configured to contact the bearings. The crank can also include one or more bushings that are inserted into the chamber holes and provide bearing surfaces for the journals. In some embodiments, the crank extends from one or more chamber side walls and does not extend through the chamber floor.

[0022] In a CPM, the crank is configured to raise and lower the slide by rotating the shaft. Because a portion of the shaft (such as a curved or asymmetrical portion) engages and raises the slide, this portion can have a coating or surface treatment that reduces friction with the slide. Alternatively, the coating or surface treatment can increase friction or strengthen engagement, allowing the crank to apply a biasing force to the slide. In some embodiments, the shaft forms a closed loop, which can be rectangular or another shape.

[0023] As used herein, "slide" refers to any sample holder, support, or substrate having at least one substantially flat surface for a biological or chemical sample. Thus, a slide can be a carrier, test tube, chip, array, or disk capable of supporting at least one sample. Typically, a slide has a first major slide surface and a second major slide surface.

[0024] The sample can be placed on the slide in a variety of ways. In some embodiments, the sample is a biological sample, such as a layer or section of skin, tumor, or other tissue. The tissue or other sample can be preserved in an embedding medium, such as formaldehyde or paraffin. Samples in paraffin or other embedding medium can be subjected to a step, such as deparaffinization, to remove the paraffin that covers and / or permeates the sample (i.e., the paraffin that covers and / or permeates the sample).

[0025] The term "conduit" generically encompasses any structure configured to define a flow path for fluid to travel from one point (e.g., the inlet of the conduit) to another point (e.g., the outlet of the conduit), although a conduit can also deliver fluid to intermediate points. A conduit can be flexible, rigid, or somewhat or partially both. For example, a conduit can be a length of tubing, a short fitting, or a manifold with multiple inlets and / or outlets (i.e., multiple inlets or multiple outlets, or both), and can be formed from plastic, metal, silica, and other materials.

[0026] The present disclosure relates to capillary processing modules (CPMs), including capillary staining modules or other modules that perform one or more processing steps described herein. The present methods and devices generally use the CPMs described herein. The CPM includes a chamber that receives a slide and performs one or more processing steps on the slide. The chamber can be of any desired shape or size and can be formed from any desired material. In some embodiments, the chamber is substantially rectangular, having long and short sides. The CPM can also include a chamber inlet that introduces a fluid (such as a liquid containing a reagent) into the chamber for processing a sample. In some embodiments, the chamber inlet is on the long side. In some embodiments, the long side is about three times as long as the short side. The chamber can be open at the top, and the CPM can include a chamber lid that closes the chamber. The chamber can have one or more sections. In some embodiments, the processing area is located in the first section and the crank is located in the second section.

[0027] In some embodiments, the capillary treatment module is formed from metal, polymer, or composite materials. For example, the chamber can be machined from stainless steel, and the treatment area can be polished to remove machining marks and achieve a very smooth surface. In some embodiments, the chamber or treatment area surface is then treated for surface hardening (e.g., Kolesterized) and then electropolished to remove treatment residues. The surface can then be treated to provide corrosion wear resistance, chemical inertness, and anti-adhesion properties. The CPM preferably has sufficient hardness to withstand scratches and the buildup of staining residues on its bottom surface, due to the very high hardness and sharp edges of many glass slides.

[0028] In some embodiments, the CPM is made from a stainless steel alloy (such as SMO254) that has been treated with a surface hardening process, such as galvanizing, to resist scratches. In some embodiments, the surface of the CPM has an inert, corrosion-resistant coating, such as an amorphous silicon, oxygen, and carbon barrier. The coating can be selected to provide one or more of corrosion wear resistance, chemical inertness, and anti-adhesion properties. For example, in some embodiments, the coating can be an inert, corrosion-resistant coating, such as Dursan. Such a coating can prevent or reduce residue, such as tissue and stain residue, remaining on the bottom surface of the CPM. The coating can be applied to the surface of the CPM by any suitable technique, such as chemical vapor deposition (CVD).

[0029] As used herein, "automated" is defined as steps performed substantially by mechanical, computer, and / or electronic means (i.e., mechanical, computer, and / or electronic means), but does not exclude any steps of human intervention, such as manually interchanging one of the features or steps described. As used herein, automated sample processing equipment also refers to an automated staining equipment or other automated equipment that performs one or more steps of sample processing.

[0030] In some embodiments, slides are automatically inserted into a CPM for processing, and then removed from the CPM after one or more processing steps have been performed within the CPM. Slide insertion and / or removal (i.e., insertion or removal, or both) can be performed, for example, by a slide gripper on a robotic arm, and thus the apparatus and method can further include an automated system for moving individual slides. The slides can be raised by a crank on the CPM to an angle (e.g., a fixed angle of 11 degrees), allowing for automatic removal of the slides by the slide gripper without mechanical tolerances of the slide gripper or robotic arm making slide removal impractical or impossible.

[0031] In some embodiments, the present methods and devices are advantageous in terms of slide handling capabilities. The chamber can be opened to accommodate a slide and closed after the slide is inserted. The chamber provides a space (i.e., a recess) to accommodate a gripper that holds the slide by its end label as it is inserted into the chamber. The CPM also generates agitation for the slide and has features to urge the slide to a desired position within the chamber, such as one or more catches in the X, Y, and / or Z directions (i.e., the X, Y, and / or Z directions). Rotation of a crank can urge the slide in the Y direction toward the end of the chamber. The CPM can have one or more guide pins to urge the slide in the X direction. In some embodiments, camera teaching points are included to facilitate automation and machine learning.

[0032] The present apparatus and method can raise and lower a slide or portion of a slide using a crank positioned within the CPM. The crank can be of any design suitable for converting motion from one dimension or direction to another, particularly converting rotational motion to linear motion. The crank can be configured to convert rotational motion to linear motion in a direction substantially perpendicular to the major surface of the slide. FIGS. 1A-1D provide a conceptual illustration of how a crank can be configured to raise and lower a slide 102. The crank includes a shaft 106 configured to rotate on an axis 107, with a bent portion 108 adapted to engage and disengage from the slide 102. FIGS. 1A and 1C provide views from the long edge of the slide 102, while FIGS. 1B and 1D are views from the short edge of the slide 102. FIGS. 1A and 1B show the slide 102 in a lowered position relative to the chamber floor 104 of the CPM. In FIGS. 1C and 1D, the slide 102 is elevated relative to the chamber floor 104 of the CPM by rotation of the shaft 106. The shaft 106 has a bent portion 108 that engages and elevates the slide 102. The shaft 106 is configured to rotate about an axis that extends horizontally in the plane of the figure. In FIG. 1D, the shaft 106 has rotated approximately 180 degrees, or half a turn, so that the bent portion 108 is above the chamber floor 104 and engages or elevates the slide 102. Rotation can also control the bias in the X direction (long side) by the vertical sidewall of the crank. As shown in FIG. 1B, the bent portion can be positioned within a recess 109 of the CPM, where the bent portion 108 can extend below the plane of the chamber floor 104 and / or the axis of rotation of the shaft 106 (i.e., the plane of the chamber floor 104, the axis of rotation of the shaft 106, or both). The chamber floor 104 is separated from the recessed floor 109 by a sloped floor 111 .

[0033] 1A and 1C also illustrate how a capillary gap can be formed and destroyed between the slide 102 and the chamber floor 104. In FIG. 1A, there is a capillary gap with a height defined by the height of the slide supports 117 and 118 on which the slide 102 rests when in the lowered position. As used herein, a "capillary gap" refers to a distance between two surfaces sufficient to allow capillary action between those surfaces. Capillary action (also called capillary action or wicking) is the flow or movement of a liquid within a narrow space due to intermolecular forces between the liquid and a solid surface. In some embodiments, the CPM is configured to provide a capillary gap of about 0.01 mm to about 0.3 mm, alternatively about 0.07 mm to about 0.1 mm, and / or a capillary gap volume of about 10 μL to about 300 μL, alternatively about 80 μL to about 100 μL, between the chamber floor and the slide in the lowered position (i.e., provide a capillary gap of about 0.01 mm to about 0.3 mm, alternatively about 0.07 mm to about 0.1 mm, or provide a capillary gap volume of about 10 μL to about 300 μL, alternatively about 80 μL to about 100 μL, or both). The sample attached to the slide faces downward, and the capillary gap is defined by the geometry of the slide and the CPM. More specifically, the sample is attached to a first major surface of the slide, and the method includes inserting the slide into the chamber so that the first major surface of the slide bearing the sample faces the chamber floor. The shape of the slide, the geometry of the CPM, the surface properties of the material, the rate of slide elevation, the angle of the slide, and the properties of the applied fluid(s) are factors that control the behavior of the fluids applied to the sample in the CPM. In FIG. 1C, the slide 102 is in the elevated position, and the capillary gap is absent because it has been broken by operating a crank to raise the slide 102 and increase its distance from the chamber floor 104. The slide 102 does not contact the slide support 118, but may remain in contact with the slide support 117 or may contact another part, such as the chamber floor 104 or the back wall of the chamber.Destroying the capillary gap while still maintaining control of the processing fluid and allowing the capillary gap to reform is a significant advantage. Furthermore, using a crank to raise and lower the slide after liquid application allows for mixing of the reagents, allowing the use of small volumes of liquid reagents, such as 50 μL to 150 μL, without localized depletion of the reagent. The slide can be raised to a specific height and / or angle (i.e., a specific height and / or angle) in a controlled manner at a certain speed, allowing mixing of the reagents in the applied fluid and buffer to be performed with a small volume of reagent, such as 90 μL, and a larger volume of buffer, such as 2000 μL. If mixing can be performed during the wash steps of the staining protocol, washing efficiency is increased.

[0034] 2A-2D show another embodiment of the present apparatus and method, in which bent portion 128 has a width greater than the width of slide 122 and the axis of rotation is above slide 122. In this manner, the crank is configured to receive or hold slide 122 within bent portion 128. In this configuration, the CPM can raise and lower slide 122 over a wider angular range.

[0035] FIG. 3 provides a perspective view of a CPM 140 according to the present disclosure. The CPM includes a housing 141 and a lid 143, which form a closed chamber when the lid 143 is in the lowered position. A shaft 146 extends through a bearing hole into a chamber defined by the chamber floor 144 of the housing 141 and the lid 143. The housing 141 has a recess 149 that accommodates a bent portion 148 of the crank when the crank is in the lowered position, allowing the shaft 146 to rotate until the bent portion 148 is positioned within the recess 149 and no longer extends above the chamber floor 144. The shaft 146 is supported within the bearing hole by bearings 151, 152, which enable the shaft 146 to rotate. The housing 141 has a sloped floor 153 that slopes from the chamber floor 144 into the recess 149.

[0036] The use of a crank offers a less readily apparent advantage in that it facilitates the creation of a chamber that is sealed or closed from the surrounding environment. The device avoids the use of lifting mechanisms or power lines that would otherwise intrude or travel into the CPM chamber from the external environment. Also, the power source providing the lifting force does not need to be located within the CPM chamber, nor does it need to use magnetic forces to drive the movement. If the mechanism passes through the bottom of the CPM, that bottom must be adequately sealed to avoid leaks and potential damage to the chamber. Furthermore, glass residue, debris, and liquids can enter the lifting mechanism at the bottom and potentially cause damage. The device avoids these issues by locating the crank on the sidewall of the chamber. The device uses a relatively simple mechanical rotation to control the elevation of a slide to a desired height within the CPM. A tangential force can be used to actively move the slide, either biasing or unbiasing it, depending on the design and direction of rotation. Additionally, the device uses the crank and CPM wall(s) to bias the slide into the desired position for processing, reducing or eliminating the need for precision when inserting the slide into the processing position by the robot.

[0037] 4 shows a different design used in the present apparatus and method. The crank has two ridges 154, 155 between the shaft 156 and the bent portion 158. The ridges 154, 155 are spaced a distance apart, allowing the slide (more specifically, the short side of the slide) to fit between them. This design provides more control over the X-direction movement of the slide as the crank raises the slide.

[0038] 5A, 5B, and 5C illustrate another embodiment of the present apparatus and method. A slide 202 is raised and lowered by a crank in a CPM. The crank has an asymmetrical portion 208 that engages with the slide 202. The asymmetrical portion 208 can be created by cutting out or omitting a portion of the shaft 206. In some embodiments, the shaft has an asymmetrical portion that engages with the slide, such as an elliptical, oblong, or eccentric shaft. The shaft 206 is supported by a bearing 211 in the side wall 205 of the chamber 201. The chamber 201 is closed by a chamber lid 212, forming an isolated space for processing the slide 202. The shaft 206 is configured so that the slide 202 does not physically contact the chamber lid 212, even when the slide 202 is raised to its uppermost position. A heater 213 is in thermal contact with the chamber lid 212. As shown in FIG. 5A, the heater 213 is a plate that covers substantially the entire area of ​​a first side of the chamber lid 212. A heater may also be provided at the bottom of the chamber 201. The heater 213 may provide substantially uniform heat to the chamber lid 212 and the chamber 201. The chamber 201 has a chamber floor 204, and when the shaft 206 and asymmetric portion 208 are rotated so that the slide 202 is in the lowered position (shown generally in FIG. 5A ), the slide 202 and the chamber floor are separated by a small gap. This gap may be provided by supporting the slide a small distance above the chamber floor 204 with one or more supports extending from the chamber floor 204. The chamber 202 may also include a sloped floor 214 extending from the chamber floor 204 toward the shaft 206 to a concave floor 215, such that the chamber 201 has a recess 216 that may accommodate the shaft 206 or a portion thereof. The sidewalls 205 join to the chamber floor 204 at curved corners 218. Other surfaces of the chamber 201 may also join the sidewall 205 at curved corners 219, which may have a different curvature or other characteristics than the corners 218. The curved corners facilitate complete removal of processing fluids from the chamber 201.In some embodiments, the CPM can have one or more chamber sidewalls that are joined to the chamber floor by curved corners having a radius of curvature of about 0.5 mm to about 5 mm.

[0039] 5C shows a cross section of the crank with asymmetric portion 208 between bearings 211, 221. The crank has a drive end 222, which has one or more features that engage a drive that rotates the crank. Drive end 222 engages a gear 223, which can be rotated by a drive chain 224 or other actuation mechanism. Drive end 222 and gear 223 may have reciprocal features or may be otherwise fastened. To ensure that heat or fluid is not lost from the chamber and to maintain the desired humidity level, a compression spring 225 is positioned between gear 223 and drive end 222 to apply a suitable force (e.g., approximately 2.6 N), thereby sealing chamber 201 from the ambient environment.

[0040] The present method and apparatus are advantageous in that they provide a defined staining area based on the slide position and liquid position within the chamber. The staining area may also be defined in part by a label on the bevel, the slide edge, and / or on the slide (i.e., on the bevel, or the slide edge, or on the slide, or all of these).

[0041] 6A-6F show various elements of another embodiment of the present apparatus and method, specifically a chamber 301 of a CPM. A slide 302 is positioned within the chamber 301, which has a chamber floor 304 and a sloped floor 314. Supports 317, 318 support the slide 302 a short distance above the chamber floor 304. The chamber 301 defines a slide space capable of accommodating the slide 302. The slide space has a chamber length L C and chamber width W C In some embodiments, L C is about 74 mm to about 106 mm (for example, about 90 mm), and W CThe chamber 302 has a processing length L of about 24 mm to about 36 mm (for example, about 30 mm). P and processing width W P A processing area A defined by P It is also possible to define a processing area A P is the area covered by the processing fluid spreading between the slide 302 and the chamber floor 304 due to capillary forces. P is about 42 mm to about 54 mm (for example, about 48 mm), and W P is about 26 mm to about 32 mm (e.g., 29 mm), and / or A P is about 1000mm 2 ~Approx. 1800mm 2 (For example, about 1400 mm 2 ) (i.e., L P is about 42 mm to about 54 mm (for example, about 48 mm), and W P is about 26 mm to about 32 mm (e.g., 29 mm), or P is about 1000mm 2 ~Approx. 1800mm 2 (For example, about 1400 mm 2 ) or both). Processing width W P is substantially the same as the slide width Ws because the capillary gap is defined by the surface of the slide. P is defined by the length of the chamber floor 304 since the capillary gap is broken by the sloped floor 314.

[0042] The present method and apparatus also have excellent liquid dispensing capabilities and features, such as a port that allows liquid to be added to the chamber without opening the chamber lid. The CPM can have one or more inlets configured to accommodate the tip position of a dispenser, such as a pipette, including the tolerance of the dispenser. The inlets can be configured to prevent liquid trapping and to connect to the capillary gap formed by the slide and chamber floor. In some embodiments, the inlets have a rounded area and / or a volume (i.e., a rounded area and / or a volume) below the slide residing in the chamber. In some embodiments, the liquid dispensing feature has one or more holes in the chamber floor or side, which can be connected to a conduit that delivers liquid, such as a wash buffer, from the outside. For example, such holes can be located in a humidity reservoir area or another area of ​​the chamber floor. The CPM allows for faster dispensing of liquid into the processing area. The CPM is configured to create a capillary gap between the slide and the chamber floor, more particularly in a flat processing area. For example, the CPM can have three knobs (or a different number of knobs) extending from the chamber floor, and the slide rests on those knobs. The height of the knobs establishes the height of the capillary gap. The CPM can also include other features that facilitate dispensing fluids into the processing region of the chamber using capillary forces. In some embodiments, pipette teaching points are included to facilitate automation and machine learning.

[0043] FIG. 6B is a cross-sectional view along the line indicated in FIG. 6A, illustrating how processing fluid can be dispensed onto slide 302 in the elevated position. Slide 302 can be elevated by a crank or another mechanism described herein. A fluid dispenser 320 is inserted into chamber inlet 322. In some embodiments, fluid is dispensed while the slide is in the elevated position or while the slide is being lowered from the elevated position. In FIG. 6A, chamber inlet 322 is located along the long side of chamber 301, but it may also be located on the short side in addition to or instead of the long side. Fluid can be dispensed while the slide is in the lowered position, and the fluid is expelled from the inlet by capillary forces during dispensing. If fluid is dispensed while the slide is elevated, the slide is lowered after dispensing to spread the fluid across the processing area. Before or after fluid dispensing, slide 302 can be elevated to a height or angle sufficient to avoid or break capillary forces between slide 302 and chamber floor 304. Some fluid from dispenser 320 may remain at inlet 322, while some is partially pushed down slide 302 by the dispensing pressure. As slide 302 descends, the fluid experiences capillary forces that distribute the fluid across the processing area, and the fluid moves toward the end of the slide that is not raised by the crank. This end may be the end with chamber outlet 326. As the crank gradually lowers slide 302, the fluid spreads toward the end of slide 302 that has been raised and is now descending. The fluid spreads across chamber floor 304 due to capillary forces, and where sloped floor 314 begins, spreading is substantially stopped or inhibited because the gap between slide 302 and sloped floor 314 is large enough to avoid capillary forces.

[0044] In some embodiments, the fluid is dispensed as a series of droplets, and the fluid can begin to spread across the processing area until all of the fluid has been dispensed, but some of the fluid should remain at the inlet as each subsequent droplet is added.

[0045] The rate of dispensing and the rate of spreading over the processing area should be balanced, and dispensing should be done at an appropriate speed or rate. The fluid should not be dispensed so quickly that some of it splashes or flows onto the top surface of the slide. In some embodiments, the rate at which the fluid is dispensed into the inlet is based on the liquid class of the fluid. Examples of dispensing rates for various liquid classes include 0.1 mL / s to 1 mL / s for liquids with a viscosity similar to water (approximately 1 centipoise at 20° C.) and as low as 0.01 mL / s for liquids with higher viscosities, such as in situ hybridization buffers.

[0046] In the CPM shown in Figures 6A and 6B, the first chamber inlet 322 has a substantially flat inlet wall 323 and a substantially flat inlet floor 325. The CPM also has a second chamber inlet 324 that has a sloped inlet wall and / or a sloped inlet floor (i.e., sloped inlet wall and / or sloped inlet floor). The inlet 324 is also shown on the long side of the chamber 301, but could also be located on a short side, such as the side closest to the outlet 326. CPM embodiments can have each of these inlet designs, or can have two (or more) inlets of the same design. The chamber inlet design can be selected based on when and how much fluid is desired to be drawn into the capillary gap as the slide is lowered.

[0047] 6C and 6D show the chamber inlet 322 in more detail. The flat design of the chamber inlet 322 is configured to form a large drop of dispensed fluid at the floor of the inlet. This drop is constrained from spreading. The dimension φ (e.g., 4.5) and the inlet width W I(e.g., 4.5 mm) is selected to push the fluid to the capillary surface. The inlet wall 323 is joined to the inlet floor 325 by an outwardly curved corner 328, and this curvature has a radius R1 (e.g., R1 can be 1.45 mm). In some embodiments, R1 is selected to ensure that substantially no fluid remains within the chamber inlet once it is captured by the increasing capillary force of the descending slide. D1 (e.g., 6.3 mm) is the distance from the far wall to a small slide (such as a 24.43 mm wide slide), and this distance corresponds to the distance the fluid needs to reach. The chamber 301 has side walls that are joined to the chamber floor by an outwardly curved corner 329 with a radius R2 (e.g., R2 can be 0.8 mm). D2 (e.g., 4.47 mm) is the distance from the chamber wall to the edge of the slide when closest to the inlet. D2 can be selected to ensure that the dispenser does not contact the edge of the slide when the slide is closest to the inlet. φ, W I , R1, D1, R2, and D2 can have dimensions other than the exemplary dimensions set forth above.

[0048] The flat design of the chamber inlet on the long side of the chamber 301 can have several advantages, including less sensitivity to whether the slide is biased or not, fluid does not end up on top of the slide, and the flat bottom of the chamber inlet makes it less sensitive to the X, Y, and Z tolerances of the dispenser.

[0049] 6E and 6F show the chamber inlet 324 in more detail. The angled design of the chamber inlet 324 is configured to reduce or prevent droplet formation at the inlet. The chamber inlet 324 has a wall 330 that forms an angle A with the chamber floor 304 (e.g., angle A of 100 to 150 degrees, or about 120 degrees), thereby reducing or preventing significant amounts of fluid from adhering to the wall 330.

[0050] 7A, 7B, and 7C show another embodiment of the present apparatus and method. A slide 402 is placed in a chamber 401. A chamber floor 404 forms a capillary gap with the slide 402 when the slide is in the lowered position, and fluid spreads throughout the capillary space due to capillary forces. The chamber 401 has a sloped floor 414 between the chamber floor 404 and a recessed floor 415. Because there is no capillary force in the larger gap between the sloped floor 414 and the slide 402, fluid does not spread widely over the sloped floor 414. The CPM includes a crank having a shaft 406 and an asymmetric portion 408 positioned above the recessed floor 415 and below the plane of the chamber floor 404. In FIG. 7B, D R is the height difference between the chamber floor 404 and the recessed floor 415. In some embodiments, D R is about 1.5 mm to about 4.5 mm, or about 3 mm. Chamber 401 has bearing holes 432 and 434 on each of its long sides. Bearing hole 432 is a blind hole, while bearing hole 434 is a through hole that allows shaft 406 to contact an operating mechanism outside chamber 401. Shaft 406 is rotatably held by bearings 433 and 435.

[0051] In some embodiments, the chamber floor of the CPM has a tongue 444 that allows the CPM to accommodate a larger volume of fluid, retaining fluid below the slide while reducing the risk of contamination from or damage to the crank. The tongue also facilitates fluid evacuation through the capillary gap while preventing liquid from reaching the crank. The tongue prevents fluid leakage as a result of capillary action, fluid movement away from the sides, radiuses on the sides, radiuses on the guide pin X, and the module angle. Exemplary dimensions for the tongue include a tongue area of ​​approximately 20 mm x 20 mm. The chamber can also have a humidity reservoir 460, which reduces evaporation from the reagent and acts like a vapor sacrifice mechanism. The humidity reservoir 460 can be filled with deionized water (DI) or another liquid by a pipette or dispenser, and can be emptied when the chamber is opened and between slide processing by a pipettor, by heating to evaporate residual liquid, or by tubing such as a tube at the bottom. Periodic emptying is desirable to avoid overfilling the reservoir or overflowing with residual liquid.

[0052] The CPM of Figures 7A, 7B, and 7C controls the position of slide 402 in several ways. In the Y direction, the desired positioning of slide 402 is controlled or facilitated by the action of a crank. When shaft 406 rotates counterclockwise (CCW) (as viewed from the perspective of Figure 7B), slide 402 is moved or biased toward the far end of the chamber (the end with outlet 426). The slide is desirably biased within the CPM so that it can be placed in a biased position after insertion and remain in the same position during staining. This bias allows staining reagent incubations, wash steps, and fluid removal to be performed without unstained areas of the sample, cross-contamination, or nonspecific staining. In the X direction, the desired positioning of the slide 402 is controlled or facilitated by guides 440, 441 near the outlet 426 and / or guides 442, 443 near the tongue 444 (i.e., guides 440, 441 near the outlet 426 and / or guides 442, 443 near the tongue 444). As shown in FIG. 7C, the chamber 401 includes an X-direction catch 450, a glide 452 that allows the slide to slide down slowly, and a control 454 that engages the slide in the X direction. Exemplary dimensions for the catch 450, glide 452, and control 454 include a 29 mm catch in the X direction that narrows to within 26.5 mm to control the slide position. The catch, glide, and control features ensure that the slide is accurately positioned within the chamber 401, even if it is delivered incorrectly.

[0053] In contrast to some previous slide processing devices, the present device does not require a slide carrier to maintain the slide in a desired position, and therefore, in some embodiments, a slide carrier is not present. Furthermore, some previous slide processing devices required slides to be manually inserted into a fixed position or for a slide carrier to hold the slide, such as a slide rack. Physical attachment of the slide to a rack or fixed feature may be required. The present device is unique in that the slide is not physically attached to a chamber or slide carrier, yet controlled movement of the slide within the chamber can be performed and the slide can be easily removed from the chamber and moved elsewhere by the device. Because the slide is not physically attached to the CPM, the device can accommodate a wide range of slide sizes. Thus, in some embodiments of the present device and method, the slide is not physically attached to the CPM and is not held by a slide carrier or rack. Also, different sizes of slides can be accommodated because, once inserted into the CPM, a crank biases the slide against the end wall of the chamber, thereby providing a predictable and reproducible processing length and processing area for the slide.

[0054] The present apparatus and methods also allow for individual processing of slides within a set of slides: each slide in the set can be inserted into a unique CPM, which allows each slide in the set to be raised or lowered by a unique crank action, dispensed with a unique set of fluid reagents, heated or coded to a desired temperature and / or at a desired rate, or subjected to other individually controlled steps or parameters.

[0055] 8A and 8B show a CPM with an inlet port and a movable plug that closes the port. In FIG. 8A, the CPM includes a chamber 501 into which a slide can be inserted and a crank configured to raise and lower the slide relative to a chamber floor 504. As described above, a capillary gap is formed when the slide is in the lowered position. The crank has a shaft 506 and an asymmetrical portion 508, with a sloped floor 511 leading to a recess in which the shaft 506 and asymmetrical portion 508 are located. The CPM also includes a chamber lid 512 that can be opened to allow the slide 502 to be inserted into the chamber 501 and can be closed to form an insulating space within the chamber 501. The chamber lid 512 can be connected to the chamber 501 by a hinge or other mechanism. The CPM also includes a heater 514 in the form of a plate as shown in FIG. 8A, although the heater may be in other forms, such as a wire or other resistive heating element contacting the chamber lid 512. The heater 514 can be an inductive heating element (e.g., a Peltier element or similar device) or a heater foil. The CPM can also include one or more temperature sensors (such as platinum resistance temperature detectors (RTDs) or thermocouples) that measure the temperature of the chamber lid 512, the chamber 501, or other portions of the CPM. The measured temperatures can be communicated to a temperature controller, which can adjust the heating and cooling devices based on the measured temperatures. The configuration of the chamber lid 512 and heater 514 can provide greater control over the temperature within the chamber 501.

[0056] In some embodiments, the present apparatus and methods reduce or avoid vertical temperature gradients within the chamber 501. During sample staining, it is desirable for the CPM chamber floor and chamber lid to be at substantially the same temperature because this avoids or reduces vertical temperature gradients above or around the slide. Because the slides exhibit the temperature of the CPM, controlling the CPM temperature is generally sufficient for processing methods. Horizontal temperature gradients in the processing region of the chamber can be controlled by power distribution and ambient losses to the bottom heater. Thus, the present apparatus and methods can include one or more heaters configured to heat the chamber lid and chamber bottom. Temperature control is maintained by heat transfer provided by electronic heater / cooler devices controlled through a temperature controller that controls one or more heater devices and / or one or more cooling devices (i.e., one or more heater devices, one or more cooling devices, or both). The temperature controller can be included in or in communication with the CPM controller.

[0057] In some embodiments, the CPM has a liquid outlet positioned to facilitate fluid removal from the chamber. The CPM has an outlet knob configured to concentrate fluid around it by capillary force. A beveled outlet directs fluid toward the bevel. In some embodiments, the angle of the slide relative to the chamber floor is adjusted (e.g., by rotating a crank) to agitate the fluid and facilitate fluid removal. The staining region can also be angled in the X and / or Y dimensions (i.e., the X and / or Y dimensions). For example, the staining region can be angled about the X axis at an angle of 0.1 to 10 degrees, or about 5 degrees, etc. The chamber includes a barrier, which can be horseshoe-shaped, around the front or back of the outlet to prevent fluid passage and / or air ingress (i.e., fluid passage, air ingress, or both). The slide rests on this horseshoe, and the opening in the horseshoe allows air entry when the slide is not angled or elevated and the pump is running. This configuration ensures a controlled discharge of fluid through the outlet. When the slide is angled / elevated, most of the opening is closed, thereby increasing the effectiveness and speed of the discharge.

[0058] In some embodiments, the slide is in a lowered position (not tilted) to prevent fluid from being trapped between the edge of the slide and the chamber floor. The CPM prevents liquid from being trapped in the chamber, such as by having side radiuses, beveled side cutouts, corner radiuses, back radiuses, and / or back dual supports (i.e., side radiuses, or beveled side cutouts, or corner radiuses, or back radiuses, or back dual supports, or all of these).

[0059] The CPM shown in FIG. 8A also has an outlet 526 that allows fluid to be withdrawn from the chamber 501. The fluid can be withdrawn after contacting the sample for a desired period of time, which can be a predetermined period depending on the desired assay or protocol. Fluid can be withdrawn when the slide is in the raised or lowered position, but withdrawal can be facilitated by elevating the slide for a portion of the withdrawal time to break the capillary gap. In some embodiments, the chamber floor 504 is slightly sloped toward the outlet 526 to promote movement in that direction. In some embodiments, the outlet 526 leads to a nozzle 527 to which tubing can be attached and fluidly connected to a pump or other outlet mechanism that provides suction to remove the fluid. Removal of fluid through the outlet by active suction can be accelerated by controlling the elevation of the slide to direct the fluid toward the outlet. Removing fluid by active suction without tilting or lifting the slide slows down the rate but provides greater control over the withdrawal, as the leading edge of the liquid slowly recedes until the liquid fills only the stained area. If another fluid is being applied, it is beneficial to raise the slide depending on the volume and fluid properties so that the fluid can be distributed within the CPM when the slide is lowered after application. Applying the fluid as the slide is raised provides a faster way for the fluid to spread from the chamber inlet. Then, as the slide is lowered, the fluid spreads, without having to enter and spread through a narrow capillary gap.

[0060] As shown in FIGS. 8A and 8B , the CPM also includes a plug 546 that engages a port 547 on the chamber lid 512 to insulate the chamber 501 from the external environment. When fluid is to be added to the chamber 501, a plug arm 548 on the chamber lid is actuated to move the plug 546 away from the port 547, thereby allowing a fluid dispenser to be inserted through the port 547. The port 547 is shown near the end of the chamber 501, but may be located elsewhere, such as a side location above the side chamber entrance shown in FIG. 6A . The movable plug 546 on the chamber lid 512 allows even greater control over the temperature and humidity within the chamber 501. The plug 546 may be formed from any suitable material, including rigid or flexible materials.

[0061] 8B shows a close-up view of plug 546 engaged with port 547, including the attachment of plug 546 to plug arm 548. Plug 546 has a plug stem 550 extending from plug face 552, and plug arm 548 has a cavity 549 that receives stem 550. Stem 550 and plug arm 548 have holes so that a dowel 551 can be inserted to retain plug 546 on plug arm 550. Cavity 549 has a larger cross-section than stem 550, and the hole in stem 550 is larger than dowel 551, allowing plug 546 to rotate slightly within cavity 549. This slight rotation about dowel 551, but limited by the sides of cavity 549, provides flexibility in how plug face 551 engages port 547, which has been found to facilitate the formation of a seal across port 547 to prevent heat and moisture loss.

[0062] 8C shows plug arm 548 engaging the chamber lid. Plug arm 548 presses the plug against the port with a suitable force, for example, about 1.5 N. Such force can be provided by including a spring 553 within the CPM and positioning spring 553 so that spring 553 biases plug arm 548 into a closed position (more specifically, a position where the plug seals the port). The CPM can also include an actuation mechanism, such as gear 554, that rotates to overcome the force applied by spring 553 and move plug arm 548 to an open position.

[0063] 9A-9C illustrate a technique for attaching the chamber lid 512 to the frame 560 to facilitate automatic opening and closing of the chamber. In some embodiments, the chamber lid 512 is flexibly attached to the frame 560 to allow slight movement in the X and / or Y directions (i.e., the X and / or Y directions). The frame 560 can be attached to an actuator, such as a gear, that can move the frame 560 and attached chamber lid 512 between an open and closed position. FIGS. 9B and 9C show close-up views of the attachment of the frame 560 to the chamber lid 512, where the chamber lid 512 has two receivers 561 and 562 that accommodate fasteners. The receivers 561 and 562 can be threaded to accommodate screws. The frame 512 has holes through which the fasteners pass and engage the receivers 561 and 562. The holes can include one or more washers positioned above, below, or within the holes to provide the desired fit between the fastener and the receiver. For example, lid washers 563, 564 are positioned above the holes, and lid shoulders 565, 566 are positioned within and below the holes. The lid shoulders 565, 566 have inner portions that can have substantially the same cross-section (e.g., diameter) as the holes, or can have a smaller cross-section to allow for slight movement. In FIG. 9B , the inner portion 567 of the front lid shoulder 565 has substantially the same cross-section as the holes in the chamber lid 512, while in FIG. 9C , the inner portion 568 of the rear lid shoulder 566 has a smaller cross-section than the holes. This approach has been found to allow slight movement when the chamber lid 512 is lowered into the chamber 501, thereby providing a better seal within the chamber 501. At the front of the chamber lid, a pressure of approximately 3 N can be applied to the lid shoulder 565 by the lid frame. The gap in Z direction between the lid washer 563 and the hole in the lid frame and the small diameter of the lid shoulder ensure a flexible connection in X and Y directions, allowing the chamber lid to adjust to handle the bottom flange, effectively absorbing tolerances and ensuring a good seal.At the rear of the chamber lid, the large clearance in Y and Z directions ensures that there are no loads / forces in Y and Z. The small clearance in X direction between the lid shoulder 566 and the hole in the frame 560 positions the chamber lid about the Z axis.

[0064] The present method and apparatus have various features that improve sample staining in a CPM. The time, temperature, and / or concentration (i.e., time, temperature, and / or concentration) of reagents used to stain samples on slides can be shortened, reduced, or better controlled. CPMs allow for easy agitation of reagents after they are dispensed, avoiding reagent depletion and the formation of bubbles that can render portions of the sample unreactive. CPMs can also provide improved control over humidity within the chamber and / or create closed compartments with high relative humidity (including up to 100% rH) while preventing condensation of fluids within the compartment. In some embodiments, CPMs have a reservoir in the chamber floor, allowing for better humidity control. Humidity can also be adjusted and selected through process control.

[0065] Figure 10 shows a capillary treatment module equipped with a cooling device. To control dyeing and other processes, it is desirable for the present CPM to have a consistent, repeatable temperature profile throughout the process. This includes control of the ramp-up and cool-down (rate of temperature increase and decrease). Generally, the time it takes to ramp up and cool down decreases when the thermal capacity of the CPM is reduced, such as by reducing its mass. In some embodiments, the treatment area (e.g., the dyeing area) of the CPM is ramped from 37.0°C to 95.0°C in 200 seconds or less, or 150 seconds or less, or 120 seconds or less, and / or cooled from 97.0°C to 35.0°C in 900 seconds or less, or 800 seconds or less, or 700 seconds or less (i.e., ramped from 37.0°C to 95.0°C in 200 seconds or less, or 150 seconds or less, or 120 seconds or less, or cooled from 97.0°C to 35.0°C in 900 seconds or less, or 800 seconds or less, or 700 seconds or less). The ramp-up time is generally related to heat capacity, heater power, and losses to the surrounding environment. The cooling is generally related to heat capacity and active cooling, such as airflow or forced convection. The bottom of the CPM is cooled primarily by cool ambient air from a blower or other cooling device, and the chamber lid is cooled primarily by conduction to the bottom of the CPM.

[0066] In FIG. 10, the CPM includes a chamber 601 having a chamber floor 604, a sloped floor 611, a chamber lid 612, and a heater 614, similar to that described with respect to the CPM of FIG. 8A. An exhaust 626 allows fluid to be withdrawn from the chamber 601 and leads to a nozzle 627 to which tubing can be attached. In FIG. 10, the chamber lid port 647 is open but can be closed by a chamber lid plug. A blower 650 is disposed within the CPM housing 651 and is preferably positioned opposite the chamber floor 604 to provide cooling air or other gas across the chamber 601. Frames can be positioned around the chamber 601 to define a flow path for cooling gas around the chamber. For example, a lower chamber frame 652 and an upper chamber frame 653 are positioned to define a flow path for cooling gas to travel from below the chamber 601 to the heater 614. This also facilitates a reproducible and consistent temperature profile around the chamber 601.

[0067] Temperature sensors can be located on one or more of the chamber lid 612, the heater, the chamber floor 604, or other portions of the CPM. As described above, the present apparatus and methods can also include a temperature controller and / or a CPM controller (i.e., a temperature controller or a CPM controller, or both) that receives temperature measurements from the temperature sensors and operates heating and / or cooling devices (i.e., heating and / or cooling devices) (e.g., blowers). The heating and / or cooling devices (i.e., heating and / or cooling devices) can be operated in an on / off or fast / slow manner to regulate the temperature of the CPM or portions thereof.

[0068] 11 shows one embodiment of a chamber lid sensor. A sensor 702 contacts a heater 708 on the surface of a chamber lid 710 via contacts 704. The sensor 702 may be surrounded by a cover 706 that is separated from the sensor 702 by a height HC.

[0069] The present apparatus and method have an advantageous closed-compartment design. Several features of the CPM contribute to the closed-compartment design. For example, some embodiments of the CPM are sealed at the crank bushing, as shown in FIG. 3B. Some embodiments of the CPM are configured with a sealing closure for the port, as shown in FIG. 8B. The CPM can be designed so that the port plug has a flat overlap (in the XY plane) with the chamber lid. As shown in FIG. 8B, the port plug can be spring-biased in the Z dimension to ensure close contact with the port (either directly or via a plug arm attached to the port plug), and the port plug can also be configured to be floatingly suspended in the Z dimension. Some embodiments of the CPM are configured so that the chamber lid is in close contact with the chamber. The CPM can be designed so that the chamber lid has a flat overlap (in the XY plane) with the chamber. As shown in FIGS. 9A-9C, the chamber lid can be spring-biased in the Z dimension to ensure close contact with the chamber (either directly or via a frame attached to the chamber lid), and the chamber lid can also be configured to be floatingly suspended in the Z dimension.

[0070] The present apparatus and method have an advantageous thermal design. The method and apparatus reduce or minimize horizontal temperature gradients through heater power distribution, and no vertical gradients occur when the bottom and lid have the same temperature. In some embodiments, the processing region has a temperature gradient within ±0.5°C (at the bottom). In some embodiments, condensation within the CPM is prevented by maintaining all internal surfaces of the CPM within ±2.0°C of a selected temperature (e.g., 97.0°C). Several features of the CPM, such as a cooling device and temperature sensors, contribute to the advantageous thermal design. This thermal design is also implemented by the design of the heaters on the chamber lid and bottom. In some embodiments, the CPM heater has a series of layers, at least one of which is a resistive heating element. For example, the CPM heater can include a series of different layers and be a 150°C UL-certified heater. The bottom layer is adapted to adhere the heater to the chamber surface or chamber lid. An exemplary material for the bottom layer includes acrylic pressure-sensitive adhesive (PSA). A heat distribution layer, such as an aluminum film, can be placed on the bottom layer to receive heat from the resistive heating element and distribute it more evenly across the chamber surface. An insulating layer can be placed between the heat distribution layer and the heating element layer. The insulating layer should be electrically insulating while allowing heat transfer with minimal loss. The heating element layer can have one or more heater tracks with resistive heating elements, such as copper-nickel alloys, that generate heat when current is passed through them. The size and material of the heater tracks can be adapted to provide the required electrical conductivity, and the heater tracks can be designed to control the force distribution to minimize horizontal gradients. The top layer can have an adhesive with an insulating layer to protect the heater tracks. It is also envisioned that the CPM heater can have one or more additional layers as desired.

[0071] 12 and 13 illustrate exemplary advantageous heater designs. FIG. 12 illustrates a resistive heating element design for a chamber lid CPM heater. This design includes a single heated zone 720 with a heater track and four unheated zones 732, 733, 734, and 735 without heater tracks. FIG. 13 illustrates a resistive heating element design for a chamber bottom CPM heater. This design includes three heated zones 740, 742, and 744 with heater tracks. In some embodiments, the chamber lid heater has one heated zone designed for 24 V and 20 W. Unheated zones have been found to be advantageous in preventing hot spots when fixed track widths and spacings are used, thereby reducing or avoiding thermal gradients in the X or Y directions of the chamber. In some embodiments, the heater tracks in a CPM heater have substantially the same width. Sensors can be placed within the hot spot zones to limit temperature overshoot. This design also ensures a fast response of the sensor to power draw, ie, reduces the load time constant seen by a proportional-integral-derivative (PID) controller.

[0072] In some embodiments, the chamber bottom heater has three heating zones, each of which can be controlled separately. For example, one heating zone can be positioned in the staining area, and two heating zones can be positioned to compensate for mounting losses, thereby allowing for a low gradient across the staining area. In some embodiments, the heating zone 740 near the CPM outlet 726 is designed for 24V, 8.0W, thereby compensating for conductive losses in the mounting feet 727, 728 and operating at a high power density due to the limited available area. A temperature sensor can be located below the fluid inlet and can also be used to detect when fluid has been dispensed. For example, a temperature change of about 2°C or less can signal that fluid has been dispensed. Using a temperature sensor(s) to determine when the liquid reagent has been properly dispensed or expelled can be highly advantageous in that it can avoid the need for additional flow meters or sensors. The temperature sensor to detect liquid expulsion can be located at the outlet or in the conduit expelling the liquid, either inside the conduit or on the exterior surface of the conduit.

[0073] In the embodiment shown in FIG. 13, the central heated zone 742 is designed for, e.g., 24 V, 14.7 W and covers the processing area where samples can be stained or otherwise processed. Non-heated zones 743, 745 do not have a heater track and are included to prevent unwanted hot spots, particularly toward the outlet zone. A sensor (such as the sensor in FIG. 11) can be placed in or near the center of the processing area. A heated zone 744 near the label on the crank and / or slide (i.e., the crank and / or slide) is designed for, e.g., 24 V, 17.3 W to compensate for conductivity losses at the attachment point. The non-heated zone 747 is asymmetric due to losses near the crank drive. The sensor can be placed toward the humidity reservoir to control water evaporation. The wire can be placed in a notch and routed away from the sensor to prevent sensor cooling. However, the resistance of the heater track limits the distance the sensor wire can be routed.

[0074] The present methods and apparatus can result in CPM with a tightly controlled temperature profile. For example, in some embodiments, the chamber space (more specifically, the processing region) can be heated to an internal temperature ranging from 95.5°C to 98°C, alternatively 95.8°C to 97.8°C, or alternatively 96.6°C to 97.3°C.

[0075] The present methods and apparatus also provide a tightly controlled temperature profile for the chamber lid, e.g., having an internal temperature ranging from 95.5°C to 98°C, alternatively 95.8°C to 97.8°C, or alternatively 96.6°C to 97.3°C. The ability to tightly control the temperature profile of the chamber lid can serve several functions. For example, the chamber lid can operate to collect liquid and reduce humidity by setting a temperature that allows condensation on the internal surface, thus allowing the chamber lid to act like a humidity reservoir. Alternatively, the chamber lid can operate to prevent condensation on the internal surface by increasing the temperature, preventing liquid from dripping into the processing area and diluting reagents.

[0076] The method and apparatus include a heater design that reduces or minimizes heat loss. In some embodiments, the chamber bottom has a plastic support to reduce conductive losses in the mounting area. In some embodiments, the CPM includes air guides that cover the vertical sides and bottom to reduce natural convection and radiation losses.

[0077] The present apparatus and method have various process control features. In addition to raising and lowering the slide by crank action, the method can include one or more processing steps for the sample. Processing steps can be performed before, during, and / or after lowering the slide and spreading the fluid across the sample (i.e., before, during, and / or after spreading). Processing steps can also be performed before, during, and / or after raising the slide and drawing fluid from the sample (i.e., before, during, and / or after drawing).

[0078] In particular, the present devices and methods relate to the processing, e.g., chemical treatment and / or staining (i.e., chemical treatment or staining or both), of at least one biological sample, e.g., a tissue section, on a slide, and to the control of fluid reagents and temperature during processing. For example, some embodiments of the present invention relate to processing thin biological samples, e.g., tissue sections, using small amounts of processing fluids.

[0079] It is contemplated that the present methods and devices may perform one or more of the following processing steps on the sample: baking, dewaxing, target retrieval (TR), washing, cleaning, dehydration, staining, hybridization, digestion, denaturation, fixation (such as with alcohol or cross-linking), enzymatic reaction (such as with horseradish peroxidase, alkaline phosphatase), tracer, chromogen, or fluorescent dye precipitation or cross-linking reaction, etc. For example, in some embodiments, the present methods may include one or more of the following processing steps: The processing steps include baking at 75°C ± 2°C; dewaxing at 60°C ± 2°C and dissolving the paraffin or other embedding medium in a solvent, followed by washing with EtOH; TR at 97°C ± 2°C, such as with HIER in TR buffer, followed by washing with WB / DI; IHC at 37°C ± 2°C, such as by contacting the sample with Envision Flex+: pAB, Block, Linker, HRP, DAB, followed by washing with WB; counterstaining, such as by contacting the sample with DI, HTX, DI, WB; IF at 37°C ± 2°C, such as with IF: pAB, and washing with WB. In some embodiments, the method can include one or more steps of a FISH assay, such as pepsin digestion at 37°C ± 2°C; application and distribution of probes at 37°C ± 2°C; denaturation at a temperature such as 80°C ± 2°C; hybridization at a temperature such as 45°C ± 2°C; and stringent washing in SWB at a temperature such as 61°C ± 2°C. The above steps can be part of processing steps preceded or followed by washing with a wash buffer or water. In some embodiments, the method can include one or more steps of a CISH assay at 37° C.±2° C., such as blocking endogenous enzyme activity of the sample, incubation with an enzyme-linked antibody, deposition of a tracer or chromogen, visualization of the tracer with a chromogen, and other related processing steps.Washing with WB; mounting in washing buffer at 37°C ± 2°C and dehydration in EtOH; and cleaning at 37°C ± 2°C with incubation in NaClO and washing with WB, washing buffer or water.

[0080] The present devices and methods can be used in the fields of cytology, histology, molecular pathology, biochemistry, immunology, microbiology, cell biology, molecular cytogenetics, and immunohistochemistry, and thus can include one or more processing procedures used in these fields. The present methods and devices can include one or more processes or fluids for processing biological samples in immunohistochemistry (IHC), in-situ hybridization (ISH), fluorescent in-situ hybridization (FISH), chromogenic in-situ hybridization (CISH), special staining (SS), silver in-situ hybridization (SISH), microarrays (tissue, protein, RNA, DNA, PNA, LNA, etc.), and other chemical and / or biological applications (i.e., other chemical and / or other biological applications). The present methods can include processes such as deparaffinization, target retrieval, and staining, particularly for in-situ hybridization (ISH) techniques. The method can include a process of examining the sample at a molecular level, including examining DNA, mRNA, miRNA, regulatory RNA, non-coding RNA, and / or proteins (i.e., DNA, or mRNA, or miRNA, or regulatory RNA, or non-coding RNA, or protein, or all of them) that cause or are otherwise associated with the disease. Such examination can be used for histological or cytological examination.

[0081] The method, when used to stain or otherwise process a sample on a slide, can include contacting the sample with multiple different fluid reagents or wash fluids. In some embodiments, the sample is subjected to treatment processes in CPM, such as deparaffinization, washing, antigen retrieval, endogenous biotin or enzyme blocking, incubation with immunological reagents, molecular probes, secondary visualization reagents, and various chromogenic reagents, washing steps, and counterstaining. The method can include raising the slide (more specifically, a portion of the slide, such as a first end) by rotating a crank, applying a first fluid reagent, lowering the slide by rotating a crank to thereby spread the first fluid reagent across a treatment area of ​​the slide, and raising the slide by rotating a crank to thereby draw out the first fluid reagent and remove the first fluid reagent from the treatment area. In some embodiments, the slide is raised and lowered one or more additional times to mix or agitate the first fluid reagent. In some embodiments, a second fluid reagent is applied after removing the first fluid reagent. The method can include, prior to applying the second fluid reagent, raising the slide by rotating a crank, lowering the slide by rotating a crank to spread the second fluid reagent over the processing area of ​​the slide, and raising the slide by rotating a crank to extract the second fluid reagent and remove the second fluid reagent from the processing area. Similarly, the above-described process can be performed with a third fluid reagent, a fourth fluid reagent, up to 10, 12, or any other desired number of fluid reagents. Additionally, the above-described process can be performed with one or more wash fluids in place of the fluid reagents.

[0082] In some protocols for processing samples, such as ISH, the sample is dehydrated before further processing, such as by staining the sample. To obtain efficient drying or dehydration of the sample, the capillary gap can be removed or destroyed so that efficient evaporation can occur, and the lid can be raised to increase the air volume for further evaporation. The method can also include raising the slide for a time sufficient for dehydration or drying.

[0083] While the present apparatus and method are generally suitable for any slide, it is contemplated that the size and shape of the chamber can be selected for standard-sized slides. Exemplary slides include glass slides, DakoFlex, and SuperFrostPlus, each having a typical dimension of 25 mm x 75 mm. In some embodiments, the slide can have a label portion, which can be positioned on the axis of the shaft. The label portion and / or the bending portion (i.e., the label portion, the bending portion, or both) can have surface features or shapes that facilitate engagement. For example, the label portion can have a surface coating that is different from the surface of the sample portion of the slide. The surface coating can be adapted to increase or decrease friction between the label portion and the bending portion.

[0084] The present devices and methods can constitute or be included within a tissue staining device, including other components and features. By way of example, a tissue staining device can include a CPM as described herein, connected (directly or indirectly) to one or more of a controller, a pipettor for dispensing fluids, a robotic arm for grasping and moving slides, reagent vials, and devices for storing and / or moving (i.e., storing and / or moving) the reagent vials. The tissue staining device can include one or more controllers configured or programmed for operation of a crank, chamber lid, pump, heater(s), chiller(s), and / or other features of the CPM (i.e., the crank, or chamber lid, or pump, or heater(s), or chiller(s), or other features of the CPM). The controller can include hardware, software, or a combination of hardware and software for operation of the system components. The controller can include one or more software programs for processing slides with samples, such as a program having a slide staining procedure, or can be configured to receive and execute software programs. The tissue staining apparatus may also include reagent vials, devices for storing reagent vials, and / or devices for providing one or more reagent vials for use by the pipettor (i.e., reagent vials, devices for storing reagent vials, and / or devices for providing one or more reagent vials for use by the pipettor). Connections between the CPM and other components may be physical, fluidic (such as via conduits), electrical, informational (such as via wireless transmission of signals), and combinations thereof.

[0085] FIG. 14 illustrates a schematic diagram of one embodiment of an automated tissue stainer 1, including various components and features. The automated tissue stainer 1 includes a first capillary staining module 140, a second capillary staining module 140′, and a third capillary staining module 140″, although it should be understood that the number of capillary staining modules can vary. In some embodiments, the first capillary staining module 140, the second capillary staining module 140′, and the third capillary staining module 140″ can be configured for immunohistochemistry (IHC) and in-situ hybridization (ISH) applications, for example.

[0086] The staining apparatus 1 can have three levels. For example, a first, lower level I can include, for example, bulk fluid containers, waste, valves, and pumps; a second, middle level II can include, for example, multiple processing units, slide storage, robotics, and reagent vials; and a third, upper level III can include, for example, coverslippers and a control unit and communications interface. Certain elements, such as the control unit 151, can be positionable on level III to roughly allow for an eye-level display, or can be positionable on level I to avoid getting in the way of manual access to modules in level II.

[0087] As shown, the automated stainer 1 can include a slide storage unit 6 and one or more processing units, such as one or more capillary staining modules 140, 140', 140''.

[0088] The automated stainer 1 can include a sample loading station 17 where slide baskets with samples are inserted into the device for processing.

[0089] One or more slide storage units 6 are configured to store one or more slides 10, which are arranged on one or more slide baskets 20 configured to hold a plurality of side-by-side slides 10. The slide storage units 6 are configured to accommodate slides in a horizontal position, a vertical position, or another suitable position.

[0090] The automated staining apparatus 1 may further comprise a number of containers 8 of bulk fluids 8a known to those skilled in the art, such as wash solutions, buffer solutions, deparaffinization solutions, target retrieval solutions, or aqueous solutions, e.g., purified water, antibody solutions, bulk staining solutions such as hematoxylin and eosin, cleaning solutions such as DAB removal solutions, etc.

[0091] The automated staining apparatus 1 may further comprise tubing 9 connecting the bulk fluid container(s) 8 to one or more processing units 2, 140, 140', 140'', a valve 7 controlling the flow of bulk fluid 8a from the bulk fluid container(s) 8 to one or more processing units 2, 140, 140', 140'', and one or more pumps 5 configured to effect flow of the bulk fluid from the bulk fluid container(s) 8 to one or more processing units 2, 140, 140', 140''.

[0092] Additionally, the automated stainer 1 may include a waste container 11 configured to store waste fluids removed by tubing (not shown) from one or more processing units 2, 140, 140', 140''.

[0093] 14, the automated stainer 1 further comprises a slide robot 12 configured to transport one or more slides 10 or one or more slide baskets 20 in the X and Y (and Z) directions as indicated by the arrows X and Y. The slide robot 12 allows slides / slide racks to be transported between the different processing units 2, 140, 140', 140'' and the storage section 6 of the stainer 100, so that biological samples placed on the slides can be processed as desired.

[0094] 14, slide robot 12 is shown schematically lifting slide 10 from slide storage 6 along the Y direction. Additionally, as indicated by the arrow, slide robot 12 can move along the X direction, for example, to the left, to first capillary staining module 140, 140', 140''.

[0095] The slide robot 12 can grab a slide 10 from the sample drawer 17 and transport the slide rack 20 and slide 10 to any station, such as the staining module 140, 140', 140'' or the wet / dry removal module 6, for processing the sample on the slide 10.

[0096] When the slide robot 12 is positioned above the slide position in the capillary staining module, the slide robot 12 can be configured to lower the slide along the Y direction into the capillary staining module and insert the slide into the correct position within the capillary staining module, as indicated by the downward arrow along the Y direction in FIG. 14.

[0097] Additionally, slide robot 12 can be configured to position slide 10 horizontally, vertically, or at an angle between horizontal and vertical. For example, slide robot 20 can grasp or release slide 10 from loading station 17 in a horizontal orientation. As a further example, slide robot 12 can grasp or release slide 10 in a vertical orientation in pre-processing module 2 or unloading station 6. In yet another example, slide robot 12 can grasp or release slide 10 in an angled orientation between horizontal and vertical, such as in staining modules 140, 140', and 140''.

[0098] Additionally, the automated staining apparatus 1 includes a fluidic robot 14 that moves the probe 16 in the X and Y (and Z) directions, as indicated by the arrows X and Y. The fluidic robot 14 can position the probe 16 above one or more fluid containers 18, a mixing station, and capillary staining modules 140, 140', 140''.

[0099] The fluidic robot 14 can further operate the probe 16 to aspirate a portion of a reagent 18a contained in any of the reagent containers 18 and transfer and apply the portion of the reagent 18a to one or more slides 10 disposed in one or more of the capillary staining modules 140, 140', 140'' for selectively staining or treating the sample on the slide. As shown schematically in FIG. 14, one or more fluid containers 18 can be arranged in a fluid container rack 19.

[0100] Thus, the fluidic robot 14 is configured to move the probe 16 between different positions within the automated stainer 1. The fluidic robot 14 can be configured, for example, to move the probe 16 to an aspiration position, e.g., at a reagent container 18, where the probe 16 aspirates a volume of reagent 18a from the reagent container 18. Furthermore, as shown schematically by the left-pointing arrow along the X direction and the downward-pointing arrow along the Y direction, the fluidic robot 14 can move the probe 16 to a dispensing position, e.g., at a slide 10 disposed in a capillary staining module 140, 140', 140'' to selectively stain or process a sample on the slide, where the probe 16 can dispense a volume of the aspirated reagent 18a onto the slide 10.

[0101] Additionally, the capillary chambers described herein can be used in conjunction with fluid dispensing mechanisms including robotic pipettes, probes, tubing, direct dispense bottles, manifolds, and the like.

[0102] The probe 16 can be moved to a cleaning fluid container 18' before aspirating a quantity of a possibly different second fluid, and a quantity of cleaning fluid can be aspirated to clean the probe 16 before aspirating a quantity of a possible new fluid by the probe 16.

[0103] As shown schematically in Figure 14, one or more reagent containers 18, 18' can be arranged in a reagent container rack 19. Multiple reagent container racks can be configured to be individually insertable and / or removable (i.e., insertable or removable or both) to accommodate a continuous workflow, i.e., the addition or removal of reagents during the ongoing processing of slides.

[0104] Furthermore, the automated stainer 1 may further comprise a coverslipper 13 configured to place a coverslip (not shown) on top of the biological sample to be processed that is placed on the slide 10 .

[0105] As shown schematically in Figure 14, the automated stainer 1 may also include a control unit 151 including process control and input / output interfaces. A suitable input may be a keyboard and a suitable output may be a monitor, or the control unit 151 may include a touch screen display.

[0106] Illustrative Embodiments Exemplary embodiments provided by the presently disclosed subject matter include, but are not limited to, the following.

[0107] Embodiment 1. A method of processing a biological sample on a slide, the method comprising: placing the slide in a capillary processing module, the capillary processing module comprising a chamber having a chamber floor and a crank positioned to raise and lower a portion of the slide relative to the chamber floor; and rotating the crank to raise and lower the portion of the slide, wherein a capillary gap is formed between the chamber floor and the slide when the slide is in the lowered position.

[0108] Embodiment 2. The method of embodiment 1, wherein the crank raises and lowers the first end of the slide while the second end of the slide rests on the chamber floor or on a slide support disposed on the chamber floor.

[0109] Embodiment 3. The method of embodiment 1 or 2, wherein the first end of the slide is elevated so that the surface of the slide and the chamber floor form an angle of about 0.1 degrees to about 25 degrees.

[0110] Embodiment 4. The method of any one of embodiments 1-3, further comprising: supplying a quantity of a first fluid to a chamber floor; spreading the first fluid across a processing area of ​​the slide by capillary forces; and elevating the slide by rotating a crank, wherein the first fluid is drawn out of the processing area by the elevating of the slide.

[0111] Embodiment 5. The method of embodiment 4, further comprising providing no more than about 2000 μL of the first fluid.

[0112] Embodiment 6. The method of embodiment 4, further comprising repeatedly raising and lowering the slide by rotating a crank, thereby drawing out and spreading the first fluid, such as to mix the fluids, prevent localized depletion of reagents, and / or displace or remove bubbles (i.e., to mix the fluids, or prevent localized depletion of reagents, or displace or remove bubbles, or all of these).

[0113] Embodiment 7. The method of embodiment 4, further comprising raising and / or lowering (i.e., raising or lowering or both) the slide by rotating a crank and removing the first fluid from the CPM through the outlet by activating a mechanism that provides suction at the outlet.

[0114] Embodiment 8. The method of embodiment 7, wherein the slide is lowered before removal of the first fluid through the outlet is complete.

[0115] Embodiment 9. The method of any one of embodiments 1-8, wherein the slide has a first slide major surface and a second slide major surface, the sample being attached to the first slide major surface, and the method further comprises inserting the slide into the chamber such that the first slide major surface having the sample faces the chamber floor.

[0116] Embodiment 10. The method of any one of embodiments 1-9, further comprising rotating a crank to lower the slide, thereby forming a capillary gap between the chamber floor and the slide.

[0117] Embodiment 11. A capillary processing module comprising a chamber having a chamber floor and one or more chamber sidewalls, and a crank positioned to raise and lower a portion of a slide when rotated.

[0118] Embodiment 12. The capillary treatment module of embodiment 11, wherein the crank extends from one or more chamber sidewalls and does not extend through the chamber floor.

[0119] Embodiment 13. The capillary treatment module of embodiment 11 or embodiment 12, wherein the crank has a bent portion positioned to engage and disengage from the slide as the crank rotates.

[0120] Embodiment 14. The capillary treatment module of any one of embodiments 11-13, wherein the crank has an asymmetric portion positioned to engage and disengage from the slide as the crank rotates.

[0121] Embodiment 15. The capillary treatment module of embodiment 14, wherein the crank has a drive end that engages the gear, and wherein one or more springs are positioned to apply a force from the gear to the drive end such that the chamber is sealed from the ambient environment.

[0122] Embodiment 16. The capillary treatment module of any one of embodiments 11-15, further comprising a cooling device positioned outside the chamber below the chamber floor.

[0123] Embodiment 17. The capillary treatment module of any one of embodiments 11-16, further comprising an outlet in the chamber and an exhaust mechanism fluidly connected to the outlet.

[0124] Embodiment 18. The capillary treatment module of embodiment 17, wherein the capillary treatment module is adapted to minimize residual fluid in the chamber after draining.

[0125] Embodiment 19. The capillary treatment module of embodiment 17, further comprising a barrier surrounding a portion of the outlet.

[0126] Embodiment 20. A capillary treatment module according to embodiment 19, wherein the barrier surrounds the front of the outlet such that an opening in the barrier allows access to the outlet when the slide is in the lowered position.

[0127] Embodiment 21. The capillary treatment module of any one of embodiments 11-20, further comprising a chamber lid movably connected to the chamber, the chamber lid being movable between an open position and a closed position.

[0128] Embodiment 22. The capillary processing module of embodiment 21, further comprising a fluid port on the chamber lid and a port plug configured to seal the port.

[0129] Embodiment 23. The capillary treatment module of any one of embodiments 20-22, wherein the chamber lid is attached to a frame configured to move the chamber lid between an open position and a closed position.

[0130] Embodiment 24. The capillary processing module of embodiment 23, wherein the chamber lid is attached to the frame by a plurality of fasteners positioned along a major axis of the chamber lid, the frame allowing the chamber lid to move in angular motion about a minor axis of the chamber lid, the fasteners closer to the axis of angular motion attaching the frame to the chamber lid while allowing the amount of movement to be limited.

[0131] Embodiment 25. The capillary treatment module of embodiment 24, wherein the CPM has one or more washers within the holes in the frame, the rear washer having an inner portion with a smaller cross section than the holes.

[0132] Embodiment 26. The capillary treatment module of embodiment 22, wherein the port plug is an elastic material.

[0133] Embodiment 27. The capillary treatment module of embodiment 22 or embodiment 26, further comprising a plug arm having a port plug on an end thereof, the plug arm configured to move the port plug toward and away from the port.

[0134] Embodiment 28. The capillary treatment module of any one of embodiments 11 to 27, further comprising a heater on the chamber and / or on the chamber lid (i.e., on the chamber or on the chamber lid, or both).

[0135] Embodiment 29. The capillary treatment module of any one of embodiments 11 to 28, wherein the capillary treatment module provides control over humidity within the chamber and / or forms a closed compartment with a relative humidity of up to 100% (i.e., provides control over humidity within the chamber, or forms a closed compartment with a relative humidity of up to 100%, or both).

[0136] Embodiment 30. The capillary treatment module of embodiment 29, wherein the heater has a heater track made from a resistive heating material.

[0137] Embodiment 31. The capillary treatment module of embodiment 29, wherein the heater track is positioned to define one or more heated zones and one or more unheated zones.

[0138] Embodiment 32. The capillary treatment module of any one of embodiments 29 to 31, further comprising a temperature sensor on the heater.

[0139] Embodiment 33. The capillary treatment module of embodiment 32, further comprising a controller configured to receive temperature measurements from the temperature sensor and adjust the heater or cooling device based on the temperature measurements.

[0140] Embodiment 34. The capillary treatment module of any one of embodiments 11-33, wherein the CPM has an inlet in one or more chamber sidewalls.

[0141] Embodiment 35. A capillary treatment module according to any one of embodiments 11 to 34, wherein one or more chamber side walls are coupled to the chamber floor by a curved corner.

[0142] Embodiment 36. The capillary treatment module of any one of embodiments 11 to 35, wherein the chamber has a first recess and a second recess separated by a treatment region, the first recess having a crank shaft disposed therein and the second recess having an outlet disposed therein.

[0143] Embodiment 37. The capillary treatment module of any one of embodiments 11-36, wherein the capillary treatment module has one or more features that retain liquid below the slide in the chamber and prevent wicking of liquid from the slide.

[0144] Embodiment 38. The capillary treatment module of any one of embodiments 11 to 37, wherein the chamber is formed from case-hardened stainless steel.

[0145] Embodiment 39. The capillary treatment module of any one of embodiments 11-38, wherein the chamber has one or more surfaces formed from stainless steel alloy SMO254.

[0146] Embodiment 40. The capillary treatment module of any one of embodiments 11 to 39, wherein the chamber has one or more surfaces that are hardened by a Kolsterizing process.

[0147] Embodiment 41. The capillary treatment module of any one of embodiments 11 to 40, wherein the chamber has one or more surfaces with an inert, corrosion-resistant coating.

[0148] Embodiment 42. The capillary treatment module of any one of embodiments 11 to 41, wherein the chamber has one or more surfaces coated with a barrier of amorphous silicon, oxygen, and carbon.

[0149] Embodiment 43. A tissue staining apparatus comprising a capillary processing module, the capillary processing module comprising: a chamber adapted to receive a slide having a tissue sample thereon; and a crank adapted to rotate to raise and lower a portion of the slide such that a capillary gap is formed between the slide and a floor of the chamber when the slide is in a fully lowered position.

[0150] Embodiment 44. The tissue staining apparatus of embodiment 43, wherein the capillary processing module is adapted to allow removal of the slide.

[0151] Embodiment 45. The tissue staining apparatus of embodiment 43 or embodiment 44, further comprising one or more controllers connected to the capillary processing module.

[0152] Embodiment 46. A tissue staining device as described in embodiment 45, wherein the one or more controllers are configured or programmed for operation of a crank, a chamber lid, a pump, one or more heaters, and / or a cooling device (i.e., a crank, or a chamber lid, or a pump, or one or more heaters, or a cooling device, or all of them).

[0153] Embodiment 47. A tissue staining device as described in embodiment 45 or 46, wherein the one or more controllers include, or are configured to receive and execute, one or more software programs for processing slides according to a slide staining protocol.

[0154] Embodiment 48. The tissue staining device of any one of embodiments 43 to 47, further comprising a pipettor for dispensing fluid.

[0155] Embodiment 49. The tissue staining apparatus of embodiment 48, wherein the capillary processing module has an inlet in one or more chamber sidewalls, the inlet adapted to receive fluid from the pipettor.

[0156] Embodiment 50. A tissue staining device as described in embodiment 49, wherein one or more chamber side walls are joined to the chamber floor by curved corners.

[0157] Embodiment 51. The tissue staining apparatus of any one of embodiments 43 to 50, further comprising a robotic arm for gripping and moving slides.

[0158] Embodiment 52. The tissue staining apparatus of any one of embodiments 43 to 51, wherein the tissue staining apparatus further comprises one or more reagent vials.

[0159] Embodiment 53. The tissue staining apparatus of any one of embodiments 43 to 52, wherein the tissue staining apparatus further comprises a device for storing and / or transporting (i.e., storing or transporting or both) reagent vials.

[0160] Embodiment 54. A tissue staining device comprising a capillary treatment module according to any one of embodiments 11 to 42.

[0161] It is understood that the terminology used herein is for the purpose of describing particular embodiments and is not intended to be limiting. The terms defined have the technical and scientific meaning of the defined terms as well as those commonly understood and accepted in the art of the present teachings.

[0162] It is noted that, in light of the present disclosure, methods and apparatus can be implemented in light of the present teachings. Furthermore, the various components, materials, structures, and parameters are included merely as illustrations and examples, and not in a limiting sense. In light of the present disclosure, the present teachings can be implemented in other applications and with other components, materials, structures, and devices while remaining within the scope of the appended claims. The claims as originally filed are as follows: Claim 1: a chamber having a chamber floor and one or more chamber sidewalls; A crank positioned to raise and lower a portion of the slide when rotated A capillary treatment module comprising: Claim 2: 10. The capillary treatment module of claim 1, wherein the crank extends from the one or more chamber sidewalls and does not extend through the chamber floor. Claim 3: The crank (i) a bent portion and / or asymmetrical portion positioned to engage and disengage from the slide as the crank rotates; (ii) a drive end that engages a gear; 3. The capillary treatment module of claim 1, further comprising one or more springs positioned to apply a force from the gear to the drive end such that the chamber is sealed from the ambient environment. Claim 4: a) a cooling device positioned outside the chamber below the chamber floor; b) an outlet in the chamber and a discharge mechanism fluidly connected to the outlet; c) a barrier surrounding a portion of the outlet, preferably surrounding a front portion of the outlet such that an opening in the barrier allows access to the outlet when the slide is in a lowered position; d) a chamber lid movably connected to said chamber, said chamber lid being movable between an open position and a closed position, preferably further comprising a fluid port on said chamber lid and a port plug configured to seal said port, said port plug preferably being of an elastic material, said chamber lid optionally further comprising a plug arm having said port plug at an end thereof, said plug arm being configured to move said port plug towards and away from said port; e) a heater on the chamber and / or on the chamber lid, the heater preferably having a heater track made from a resistive heating material, the heater track most preferably positioned to define one or more heated zones and one or more unheated zones; 4. The capillary treatment module according to claim 1, further comprising at least one of the following: Claim 5: 5. The capillary treatment module of claim 4, wherein the chamber lid is attached to a frame configured to move the chamber lid between an open position and a closed position, the chamber lid preferably being attached to the frame by a plurality of fasteners positioned along a major axis of the chamber lid, the frame moving the chamber lid in angular motion about a minor axis of the chamber lid, the fasteners closer to the axis of angular motion attaching the frame to the chamber lid while allowing the amount of movement to be limited, and most preferably the capillary treatment module comprises a lid having one or more washers within holes in the frame, the rear washers having an inner portion with a smaller cross-section than the holes. Claim 6: 5. The capillary treatment module of claim 4, further comprising a temperature sensor on the heater, and optionally further comprising a controller configured to receive temperature measurements from the temperature sensor and adjust the heater or cooling device based on the temperature measurements. Claim 7: the capillary treatment module having an inlet in the one or more chamber sidewalls; the one or more chamber sidewalls are joined to the chamber floor by a curved corner; The chamber has a first recess and a second recess separated by a processing region, the first recess having a shaft of the crank disposed therein, and the second recess having an outlet disposed therein. 7. The capillary treatment module according to claim 1, comprising one or more of the following: Claim 8: The chamber comprises: Constructed from case-hardened stainless steel, having one or more surfaces formed from stainless steel alloy SMO254; having one or more surfaces that are hardened by a cholesterizing process; 8. The capillary treatment module of claim 1, comprising one or more of: having one or more surfaces with an inert corrosion-resistant coating; and having one or more surfaces coated with a barrier of amorphous silicon, oxygen, and carbon. Claim 9: 9. A tissue staining device comprising the capillary treatment module according to claim 1. Claim 10: a) one or more controllers connected to the capillary treatment module, the one or more controllers preferably including one or more of: (i) configured or programmed for operation of the crank, or chamber lid, or pump, or one or more heaters, or chillers, or all of the above; and (ii) including, or configured to receive and execute, one or more software programs for processing slides according to a slide staining protocol; b) a pipettor for dispensing fluid, the capillary processing module preferably having an inlet in one or more chamber sidewalls, the inlet adapted to receive fluid from the pipettor; c) a robotic arm for gripping and moving the slide; d) one or more reagent vias; e) Devices for storing and / or transporting reagent vials; The tissue staining device according to claim 9, further comprising: Claim 11: 1. A method for processing a biological sample on a slide, comprising: Placing a slide in a capillary treatment module according to any one of claims 1 to 8; rotating the crank to raise and lower a portion of the slide, forming a capillary gap between the chamber floor and the slide when the slide is in the lowered position; A method comprising: Claim 12: 12. The method of claim 11, wherein the crank raises and lowers a first end of the slide while a second end of the slide rests on the chamber floor or on a slide support disposed on the chamber floor. Claim 13: 13. The method of claim 11 or 12, wherein the first end of the slide is elevated so that the surface of the slide and the chamber floor form an angle of about 0.1 degrees to about 25 degrees. Claim 14: providing a volume of a first fluid, preferably about 2000 μL or less, to the chamber floor; spreading the first fluid over a treatment area of ​​the slide by capillary forces; elevating the slide by rotating the crank, wherein the first fluid is extracted from the treatment area by the elevating of the slide; Optionally, the method includes one or more of: (i) repeatedly raising and lowering the slide by rotating the crank, thereby drawing out and spreading the first fluid, such as to mix the fluid, prevent localized depletion of reagents, and / or to displace or remove bubbles; and (ii) removing the first fluid from the capillary treatment module through the outlet by rotating the crank to raise and / or lower the slide and activating a mechanism to provide suction at the outlet, wherein the slide is preferably lowered before the removal of the first fluid through the outlet is completed. The method according to any one of claims 11 to 13, further comprising: Claim 15: 15. The method of claim 11, further comprising inserting the slide into the chamber such that the slide has a first major surface and a second major surface, a sample attached to the first major surface, and the first major surface with the sample attached faces the chamber floor.

Claims

1. a chamber having a chamber floor and one or more chamber sidewalls, the one or more chamber sidewalls joining one or more sides of the chamber; a crank positioned to raise and lower a portion of a slide when rotated, the slide having a flat surface and the one or more chamber side walls having at least one bearing hole for a shaft of the crank; a capillary gap can be formed between the flat surface of the slide and the surface of the chamber floor to allow capillary action; A capillary processing module, wherein the crank has a bent portion or an asymmetric portion, or both, positioned to engage and disengage with a portion of the slide when the crank rotates, the bent portion being formed by bending the shaft of the crank so that the portion of the slide can be accommodated in the bent portion when the crank rotates, and the asymmetric portion being formed by cutting out or omitting a portion of the shaft of the crank so that the portion of the slide can be accommodated within the cut out or omitted portion of the shaft when the crank is rotated to a predetermined angle.

2. The capillary treatment module of claim 1 , wherein the crank extends from the one or more chamber sidewalls and does not extend through the chamber floor.

3. 3. The capillary treatment module of claim 1, wherein the crank has a drive end that engages a gear to rotate the crank, and one or more springs are positioned between the gear and the drive end to apply a force from the gear to the drive end so that the chamber is sealed from the ambient environment to prevent heat or fluid loss from the chamber.

4. a) a cooling device positioned outside the chamber below the chamber floor; b) an outlet in the chamber and a discharge mechanism fluidly connected to the outlet; c) a barrier surrounding a portion of the outlet, the barrier surrounding a front portion of the outlet such that an opening in the barrier allows access to the outlet when the slide is in a lowered position; d) a chamber lid movably connected to the chamber, the chamber lid being movable between an open position and a closed position, the chamber lid further comprising a fluid port on the chamber lid and a port plug configured to seal the fluid port, the port plug being made of an elastic material, the chamber lid further comprising a plug arm having the port plug at an end thereof, the plug arm being configured to move the port plug toward and away from the fluid port; e) a heater on the chamber or on the chamber lid, or both, the heater having a heater track made from a resistive heating material, the heater track positioned to define one or more heated zones and one or more unheated zones; The capillary treatment module according to any one of claims 1 to 3, further comprising at least one of:

5. 5. The capillary treatment module of claim 4, wherein the chamber lid is attached to a frame configured to move the chamber lid between an open position and a closed position, the chamber lid is attached to the frame by a plurality of fasteners positioned along a major axis of the chamber lid, the frame moving the chamber lid in an angular motion about a minor axis of the chamber lid, the fasteners closer to the axis of the angular motion attaching the frame to the chamber lid while allowing the amount of movement to be limited, the capillary treatment module comprising the chamber lid having one of the plurality of fasteners in each of a plurality of holes in the frame, each of the plurality of fasteners having a through portion with a smaller cross-section than a corresponding hole in the plurality of holes.

6. 5. The capillary treatment module of claim 4, further comprising a heater, further comprising a temperature sensor on the heater, and further comprising a controller configured to receive temperature measurements from the temperature sensor and adjust the heater or cooling device based on the temperature measurements.

7. the capillary treatment module having an inlet in the one or more chamber sidewalls; the one or more chamber sidewalls are joined to the chamber floor by a curved corner; The chamber has a first recess and a second recess separated by a processing region, the first recess having the shaft of the crank disposed therein, and the second recess having an outlet disposed therein. A capillary treatment module according to any one of claims 1 to 6, comprising one or more of:

8. The chamber comprises: Constructed from case-hardened stainless steel, having one or more surfaces formed from stainless steel alloy SMO254; having one or more surfaces that are hardened by a cholesterizing process; 8. The capillary treatment module of any one of claims 1 to 7, comprising one or more of: having one or more surfaces with an inert corrosion resistant coating; and having one or more surfaces coated with a barrier of amorphous silicon, oxygen, and carbon.

9. A tissue staining device comprising the capillary treatment module according to any one of claims 1 to 8.

10. a) one or more controllers connected to the capillary treatment module, the one or more controllers including one or more of: (i) configured or programmed for operation of the crank, or chamber lid, or pump, or one or more heaters, or chillers, or all of the above; and (ii) including, or configured to receive and execute, one or more software programs for processing slides according to a slide staining protocol; b) a pipettor for dispensing fluid, the capillary processing module having an inlet in one or more chamber sidewalls, the inlet adapted to receive fluid from the pipettor; c) a robotic arm for gripping and moving the slide; d) one or more reagent vials; e) A device for storing and / or transporting reagent vials; The tissue staining device according to claim 9, further comprising:

11. 1. A method for processing a biological sample on a slide, comprising: Placing a slide in a capillary treatment module according to any one of claims 1 to 8; rotating the crank to raise and lower a portion of the slide, forming a capillary gap between the chamber floor and the slide when the slide is in the lowered position; A method comprising:

12. 12. The method of claim 11, wherein the crank raises and lowers a first end of the slide while a second end of the slide remains on the chamber floor or on a slide support disposed above the chamber floor.

13. 13. The method of claim 12, wherein the first end of the slide is raised so that the flat surface of the slide and the chamber floor form an angle of about 0.1 degrees to about 25 degrees.

14. providing about 2000 μL or less of a first fluid to the chamber floor; spreading the first fluid over a treatment area of ​​the slide by capillary forces; elevating the slide by rotating the crank, wherein the first fluid is extracted from the treatment area by the elevating of the slide. Further comprising:

14. The method of any one of claims 11 to 13, further comprising one or more of: (i) repeatedly raising and lowering the slide by rotating the crank, thereby drawing out and spreading the first fluid, such as to mix the fluids, prevent localized depletion of reagents, and / or to displace or remove bubbles; and (ii) removing the first fluid from the capillary treatment module through the outlet by rotating the crank to raise and / or lower the slide and activating a mechanism that provides suction at the outlet, wherein the slide is lowered before the removal of the first fluid through the outlet is completed.

15. 15. The method of any one of claims 11 to 14, further comprising inserting the slide into the chamber such that the slide has a first major slide surface and a second major slide surface, the first major slide surface being the flat surface of the slide, a sample attached to the first major slide surface, and the first major slide surface having the sample facing the chamber floor.

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