WO2013090567A2

WO2013090567A2 - Immunohistochemical validation devices and methods

Info

Publication number: WO2013090567A2
Application number: PCT/US2012/069499
Authority: WO
Inventors: Jeffrey David BASSLER; Barry Michael Sandbank; Erico VON BUEREN
Original assignee: Lab Vision Corporation
Priority date: 2011-12-13
Filing date: 2012-12-13
Publication date: 2013-06-20
Also published as: WO2013090567A3

Abstract

A method for validating an immunohistochemical slide. The slide is pre-treated and stained in accordance with an immunohistochemical protocol and includes a validation portion with at least one control group. The at least one control group is configured to validate at least one parameter directed to at least one of the pre-treatment or of the staining of the slide. The method includes capturing an image of the slide and measuring a value of the at least one parameter for the control group from the image. The measured value of the at least one parameter is compared to an acceptable value. Based on this comparison, an indication of the validity of the IHC slide is generated.

Description

IMMUNOHISTOCHEMICAL VALIDATION DEVICES AND METHODS

FIELD OF THE PRESENT INVENTION

[0001] The present invention relates generally to immunohistochemical protocols and, more specifically, to devices and methods for validating a slide pre-treated and stained in accordance with an immunohistochemical protocol.

BACKGROUND OF THE PRESENT INVENTION

[0002] Immunohistochemistry ("IHC") is set of processes performed on a slide, manually and/or automatically by one or more robotics or instruments, by which the expression and localization of at least one target is detected and visualized. The target is generally an antigen (more specifically, proteins, polypeptides, peptides) having an epitope, or active site, that reacts with a detection agent. Detection agents may include a primary antibody (monoclonal or polyclonal) that specifically binds to the epitope or antigen. Various other processes may then be performed, for example, amplification, or colorization, for visualizing the detected epitope or antigen. Colorations may include, for example, fluorescence, color staining, or luminescence. The intensity of

fluorescence, color, illumination, etc. may then be used to provide quantitative data with respect to the levels of antigen present and may provide diagnostic information. As such, IHC protocols have provided particular benefit to the field of pathology where the protein expression, cell structure, and other biological markers are investigated, diagnosed, monitored, or treated.

[0003] However, the accuracy and precision of the result is determined by the IHC protocol itself. If the antibodies used in the IHC protocol are not adequately selective, then false positives may result.

[0004] Yet, even when the antibodies are sufficiently selective for the antigen of interest, there still exists that potential for error made by the laboratory technician performing the IHC protocol. Incorrect reagents, inadequate reagent concentrations, reaction timing, sample washing, and sample pre-treatment may all degrade the effectiveness of the IHC protocol. In fact, fluctuations in laboratory temperature may also affect the IHC results. Ultimately, inaccurate or inconsistent staining may result in false positives or false negatives in the antibody binding and, possibly, an inaccurate diagnosis.

[0005] One known method of attempting to provide quality control of the IHC protocol has included a plurality of synthetic controls at varying concentrations that provide, in essence, a calibration curve. In this way, the pathologist may compare a stained tissue with the plurality of synthetic controls to assess the concentration of stain. However, this known approach is configured to assess or provide an indication of a potential error in the performed IHC protocol. Therefore, there remains a need for devices and methods that are capable of validating sample preparation in accordance with an IHC protocol, prior to pathological evaluation. Such devices and methods would reduce the occurrence of pathological evaluation of improperly or poorly pre- treated, stained, or preserved samples and increase the reproducibility of the IHC protocol for making a diagnosis.

SUMMARY OF THE PRESENT INVENTION

[0006] The present invention overcomes the foregoing problems and other known shortcomings, drawbacks, and challenges associated with samples prepared in accordance with an invalid or inaccurately performed IHC protocol. While the invention will be described in connection with certain embodiments, it will be understood that the invention is not limited to these embodiments. To the contrary, this invention includes all alternatives, modifications, and equivalents as may be included within the spirit and scope of the present invention.

[0007] According to one embodiment, the present invention is directed to a method of validating an IHC slide. The slide is pre-treated and stained in accordance with an IHC protocol and includes a validation portion with at least one control group. The at least one control group is configured to validate at least one parameter directed to at least one of the pre-treatment or of the staining of the slide. The method includes capturing an image of the slide and measuring a value of the at least one parameter for the control group from the image. The measured value of the at least one parameter is compared to an acceptable value. Based on this comparison, an indication of the validity of the IHC slide is generated.

[0008] In accordance with another embodiment of the present invention, a method of validating an IHC slide pre-treated and stained in accordance with an IHC protocol includes capturing an image of the IHC slide. A parameter related to the pre- treatment control of the pre-treatment group on the IHC slide is detected and compared to an acceptable value. Based on the comparison, an indication of the validity of the IHC slide pre-treatment is generated.

[0009] Still another embodiment of the present invention is directed to a method of validating an IHC slide having a validation portion with a fixed spot and an unfixed spot. The slide is subjected to an IHC protocol that includes an antigen retrieval protocol and a staining protocol. The method includes capturing an image of the IHC slide. At least one parameter of at least one of the fixed and at least one of the unfixed spots are determined. The IHC protocol is validated based, at least in part, on the at least one determined parameter of the at least one fixed spot and the at least one determined parameter of the at least one of unfixed spot.

[0010] According to yet another embodiment of the present invention, a method of validating an IHC slide having a validation portion with a plurality of fixed spots and a plurality of unfixed spots includes determining a spectral property of at least one spot of each of the pluralities of fixed and unfixed spots. The IHC protocol is validated, at least in part, based on the spectral densities of the at least one spots of each of the pluralities.

[001 1] In accordance with one embodiment of the present invention, a method of training an apparatus for validating an IHC slide includes preparing and staining a plurality of references slides according to an IHC protocol. Each one of the plurality of reference slides includes a validation portion having at least one control. An image of the each pre-treated and stained reference slide is captured and an area corresponding to the at least one control identified. A characteristic of the at least one control is determined from each corresponding captured image and a first statistical value is calculated the characteristics of the controls from the references slides.

[0012] One embodiment of the present invention is directed to an IHC validation device. The device includes an imaging assembly comprising a light source and a light receiving device. The light source and the light receiving device define an optical path. The device further includes a slide tray having an IHC slide cradle and a calibration slide cradle. The slide tray may be positioned in a first position such that IHC slide cradle is positioned in the optical path or in a second position such that the calibration slide is positioned in the optical path.

[0013] The above and other objects and advantages of the present invention shall be made apparent from the accompanying drawings and the descriptions thereof.

BRIEF DESCRIPTION OF THE FIGURES

[0014] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments of the present invention and, together with a general description of the invention given above, and the detailed description of the embodiments given below, serve to explain the principles of the invention. [0015] FIG. 1 is a perspective view of an immunohistochemistry validation device in accordance with an embodiment of the invention.

[0016] FIG. 2 is a perspective view of an imaging assembly of the

immunohistochemistry validation device of FIG. 1 and in accordance with an

embodiment of the invention.

[0017] FIG. 3 is a cross-sectional view of the immunohistochemistry validation device taken along the line 3-3 in FIG. 1 .

[0018] FIG. 4 is a diagram of a computer system environment suitable for use with the immunohistochemistry validation device of FIG. 1 , according to an embodiment of the invention.

[0019] FIG. 5 is a flowchart illustrating a sequence of operations for

manufacturing a validation slide in accordance with an embodiment of the invention.

[0020] FIGS. 6A-6D schematically illustrate the sequence of operations of FIG. 5.

[0021] FIG. 7 is a flowchart illustrating a sequence of operations for preparing and staining the validation slide of FIG. 5 in accordance with a selected

immunohistochemistry protocol.

[0022] FIGS. 8A-8D schematically illustrate the sequence of operations of FIG. 7.

[0023] FIG. 9 is a flowchart illustrating a sequence of operations for validating the validation slide pre-treated and stained in accordance with FIG. 7 and in accordance with an embodiment of the invention.

[0024] FIG. 10 is a flowchart illustrating a sequence of operations for validating antigen removal and completion of the selected immunohistochemistry protocol in accordance with an embodiment of the invention.

[0025] FIG. 1 1 is a flowchart illustrating a sequence of operations for validating the staining procedure performed on the validation slide according to an embodiment of the invention.

[0026] FIGS. 12A and 12B are exemplary screenshots of the validation results provided by the computer in accordance with an embodiment of the invention.

[0027] FIG. 13 is a flowchart illustrating a sequence of operations for performing a training session for the immunohistochemistry validation device of FIG. 1 with a plurality of validation slides and in accordance with an embodiment of the invention.

[0028] FIG. 14 is a flowchart illustrating a sequence of operations for calibrating the imaging assembly of FIG. 2 and in accordance with an embodiment of the invention. [0029] FIG. 15 is a flowchart illustrating a sequence of operations for determining the validity of a calibration of the imaging assembly of FIG. 2 and in accordance with an embodiment of the invention.

[0030] FIGS. 16A and 16B are top elevational views of two exemplary calibration slides for use with the sequence of operations provided in FIGS. 14 and 15, in accordance with embodiments of the invention.

DETAILED DESCRIPTION

[0031] Turning now to the Figures, particularly FIGS. 1 -3, an

immunohistochemical validation device ("IHC device") 20 for validating an IHC procedure is shown in accordance with one embodiment of the present invention. The IHC device 20 includes a housing 22 enclosing various electrical and analytical instruments as described in greater detail below. A display screen 24, which may be part of a user interface 96 (FIG. 4), is in an operable position with respect to the housing 22 and is configured to provide a mechanism through which the user may exchange information with the IHC device 20, such as by touch screen capabilities.

[0032] A drawer 28 may be operably coupled to the housing 22 and is operably coupled to a slide tray 30 that extends into the housing 22 such that the slide tray 30 may slide into, and be at least partially withdrawn from, the housing 22. The slide tray 30 includes a validation slide cradle 32 and a calibration slide cradle 34 that are configured to receive a validation slide 36 pre-treated in accordance with an IHC protocol (including, for example, deparaffinization, antigen retrieval, and staining) and a calibration slide 38 (FIG. 9A), respectively, and which are described in greater detail below.

[0033] The various electrical components associated with an imaging assembly 40 and a controller assembly 42 are enclosed within the housing 22. The imaging assembly 40 is configured to acquire at least one signal related to a loaded validation slide 36 and/or the calibration slide 38, in accordance with one exemplary method as described in greater detail below. The controller assembly 42 is configured to control at least a portion of the signal acquisition.

[0034] The imaging assembly 40 may include a light source 44 and a light receiving device (illustrated herein as a camera 46), wherein the camera 46 is positioned on an opposing side of the slide tray 30 as compared with the light source 44. As shown, the camera 46 is vertically offset form the light source 44 and, as a result, a mirror 48 is included therewith. In that regard, a light path 49 may be defined as extending from the light source 44, through the slides 36, 38 (as appropriate), reflected at the mirror 48, and into the camera 46. It would be readily understood that this arrangement is but one possible configuration of the imaging assembly, and the imaging assembly is not limited to a light path that includes, or is defined by, a single mirror 48, a single camera 46, or a single light source 44. For example, the imaging assembly 40 may be operated with no or multiple mirrors so that the light path 49 may be arranged as desired. The relative positions of the camera 46 and the mirror 48 may be maintained within the housing 22 by a structural support 50. Furthermore, as the light source 44, as shown, may be positioned beneath the slide tray 30, with a debris tray 52 positioned therebetween. The debris tray 52 is configured to transmit the light emitted by the light source 44 without interfering with the optical properties of the light. The debris tray 52 may be further configured to protect the light source 44 from contaminants and/or damage. In some embodiments, the debris tray 52 may be in slidable relation with housing 22 and, optionally, may include a handle 54 such that the debris tray 52 may be at least partially removed and cleaned, as necessary, to resist interference with the transmitted light. In an alternative embodiment of the invention, the relative positions of the light source 44 and camera 46 may be reversed, in which case the debris tray 52 would be configured to protect the camera 46 from

contaminants and/or damage.

[0035] The light source 44 may generate light in the visual spectrum as well as light outside of the visual spectrum such as ultraviolet light or other wavelengths of light that elicit fluorescence in the image to be analyzed. In one embodiment, the light source 44 may be constructed as a multi-layer fiber optic panel having at least one illuminating layer, a reflective layer, and a diffusing layer. Suitable commercially- available light sources may include those that are manufactured by LUMITEX, Inc. (Strongsville, Ohio), using light emitting diodes ("LEDs") and optical fibers to evenly distribute the light. Cameras that are suitable for use with the IHC device 20 may include, for example, those that are compact, high speed, high resolution, and configured to operate at a low power. One such camera includes the commercially- available CHAMELEON digital camera from Point Gray Research Inc. (Richmond, British Columbia). While the resolution necessary for evaluation may depend, at least in part, on an area covered by a control group (as described in detail below), one exemplary example may include a resolution of greater than about 30 pixels per mm for control group diameters of about 2.4 mm. [0036] Optionally, LED inputs in the light source 44 may comprise a combination of separate monochrome red, green, and blue LEDs, which may be switched individually to produce separate color channel images to be analyzed with increased image resolution. The LED inputs in the light source 44 may comprise monochrome LEDs of a single color, such as blue, which are linked to maximizing image signal at a particular wavelength range.

[0037] Although not shown, also, one or more optical filters may be positioned within or along the light path 49 to alter at least one optical property of the light transmitted from the light source 44 and received at the camera 46 as desired. Filters may be configured to filter noise, one or more wavelengths, be polarizing, and so forth, as is known by those of ordinary skill in the art.

[0038] One of ordinary skill in the art would readily appreciate the light receiving device may include other devices, including analytical instruments, including, for example, spectrometers. Generally, the light receiving device may be any device configured to acquire at least one signal related to a color or staining of the validation slide 36 (or calibration structures of the calibration slide 38) and that may be compared with a known signal range and that is indicative of a validity of the IHC protocol (including sample pre-treatment and a staining protocol) performed on the validation slide 36.

[0039] The light source 44 and the camera 46 may be electrically coupled to a power supply 56, for example, by conductors, such as electrical cables or wires 58. The power supply 56, in turn, may include an internal power source (such as batteries) or be coupled to an external power source, such as a source of 50 or 60 Hz alternating current line power. A switch 60 may be operably coupled to the power supply 56 such that the user may power the IHC device 20 on or off as necessary. The power supply 56 may be further configured to provide power to other components of the IHC device 20. For example, a fan 100 may be coupled to the housing 22, proximate to one or both of imaging assembly 40 and the controller assembly 42 so as to maintain an operable temperature for the assemblies 40, 42. The power supply 56, or an additional power supply, may also power the controller assembly 42. The controller assembly 42 may comprise one or printed or flex circuit boards 70, hard drives 72, and a controller 74 configured to perform one or more functions and/or features of the IHC device 20 as described in greater detail below.

[0040] Referring now to Figure 4, the controller 74 may operate in a computing environment that includes one or more networked computers 76 and/or servers 78 communicatively coupled to the controller 74 by a network 80. The controller 74 may be implemented using one or more processors 82 selected from microprocessors, micro-controllers, digital signal processors, microcomputers, central processing units, field programmable gate arrays, programmable logic devices, state machines, logic circuits, analog circuits, digital circuits, and/or any other devices that manipulate signals (analog and/or digital) based on operational instructions that are stored in a memory 84. Memory 84 may be a single memory device or a plurality of memory devices including but not limited to read-only memory ("ROM"), random access memory ("RAM"), volatile memory, non-volatile memory, static random access memory ("SRAM"), dynamic random access memory ("DRAM"), flash memory, cache memory, and/or any other device capable of storing digital information.

[0041] Mass storage device 86 may be a single mass storage device or a plurality of mass storage devices including but not limited to hard drives, optical drives, tape drives, non-volatile solid state devices and/or any other device capable of storing digital information. Mass storage device 86 may include hard drive 72 and/or mass storage provided by external resources, such as devices located in a separate enclosure (not shown) or in networked computer 76 and/or server 78. A network interface 88 employs a suitable communication protocol for communicating with other computer resources over the network 80. To this end, the network interface 88 may include one or more wired port, such as an Ethernet port 90 (Figure 1 ), or a wireless device, such as an IEEE 802.1 1 transceiver, which is commonly known as Wi-Fi.

[0042] Processor 82 may operate under the control of an operating system 92 residing in memory 84. The operating system 92 may manage controller resources so that computer program code 94 embodied as one or more computer software applications residing in memory 84 may have instructions executed by the processor

82. Program code 94 typically comprises one or more instructions that are resident at various times in the memory 84 and/or the mass storage device 86 of the controller 74 that, when read and executed by the processor 82, causes the controller 74 to perform the steps necessary to execute steps or elements embodying the various aspects of the present invention. In an alternative embodiment of the invention, the program code 94 may be run by the processor 82 directly, in which case the operating system 92 may be omitted. A user interface 96 is also operatively coupled to the processor 82 of the controller 74 in a known manner. The user interface 96 may include the touch screen

24, as well as other output devices such as alphanumeric displays and other visual and/or audible indicators. The user interface 96 may also include input devices and controls, such as the touch screen 24, an alphanumeric keyboard, a pointing device, keypads, pushbuttons, control knobs, etc., capable of accepting commands or input from the operator and transmitting the entered input to the processor 82.

[0043] The controller 74 may be in communication with one or more external resources through the network interface 88, such as the networked computer 76 and/or server 78. The network resources may be also be part of a cluster or other distributed computing system. Network server 78 may be a Laboratory Information System ("LIS") server configured to provide information regarding IHC protocols and/or samples in response to queries by the controller 74 as will be described in more detail below.

[0044] To validate the IHC procedure performed on the validation slide 36 prior to pathological evaluation, the validation slide 36 includes not only the tissue sample 1 12, but at least one control group that is directed to one or more parameters corresponding to the IHC protocol, including, for example, pre-treatment and staining. Said another way, the control group is not directed to an indication used in determining a diagnosis but rather is directed to a parameter that is reflective of a quality control of the performed IHC protocol as compared with a standard operating procedure. In that regard, FIGS. 5 and 6A-6D illustrate a method of manufacturing the validation slide 36 for use with the IHC device 20 and in accordance with one embodiment of the present invention. The one or more parameters may include, for example, a spatial

characteristic or dimension (e.g., diameters, areas, and boundary detection), an optical characteristic (e.g., transmittance, reflectance, optical density, luminescence, polarization, etc.), a chemical characteristic (e.g., radioactivity), statistical comparisons (coefficient of variance or color ratios) or other such analytical measurement.

[0045] The validation slide 36 may comprise a conventional microscope slide 102 or other glass, quartz, or polymeric substrate that is suitable for transmission of light. A label 104 may be positioned on the slide 102 and include a machine readable code 106 (such as an RFID or a barcode), an indicia (illustrated as the same), and/or an identifier (illustrated as the same), one or more of which may be used for identifying the patient from which a sampled tissue was taken, a protocol performed, a date of protocol, and so forth. If necessary, the machine readable code 106 may be used by the controller 74 to identify and access such information, which may be stored locally on the mass storage device 86, or retrieved from a resource connected via the network 80. For example, the information may be retrieved from the server 78, which may be an LIS server. [0046] Because the validation slide 36 is pre-treated for use with a particular protocol, the tradename or other identifying indicator may also be included on the label, or as shown, just below the label (illustrated herein as "HER2"). Alternatively, the validation slide 36 may be manufactured for use with a selected one IHC protocol from a plurality of IHC protocols and in which an indicia, trademark, or other identifying indicator may be used by the IHC device 20 in determining an appropriate

predetermined range for comparison.

[0047] The slide 102 further includes a validation portion 108 and a sample portion 1 10 thereon. The validation portion 108 may be prepared during slide manufacture while the sample portion 1 10 is configured to receive a sampled tissue 1 12 (FIG. 8A) as explained in greater detail below.

[0048] The validation portion 108, as shown in FIG. 6A, includes at least one control group, which may be a pre-treatment control group, a staining control group, or both, wherein each control group is configured to indicate the validity of at least one portion of the IHC procedure. Each control group may include a control in singly, duplicate, triplicate, or other multiplicity as desired. As specifically shown, the validation portion 108 includes a pre-treatment control group with duplicate controls and a staining control group, also with duplicate controls. The illustrative controls are, therefore, arranged into four quadrants: a first control within the staining control group (illustrated as "U1 "), a second control within the second staining control group (illustrated as "U2"), a first control within the pre-treatment control group (illustrated as "F1 "), and a second control within the pre-treatment control group (illustrated as "F2").

[0049] For illustrative purposes only, the pre-treatment control groups F1 , F2 are described herein as being directed a sample pre-treatment step, for example, antigen retrieval. The staining control groups U1 , U2 are described herein as being directed to the staining of the validity slide 36 in accordance with the particular IHC protocol. Any combination and/or number of control groups may be used for evaluating and validating various sample pre-treatment and staining steps on a single validity slide 36. Such sample pre-treatment steps may include, for example, deparaffinization, antigen retrieval, sample mount, and so forth.

[0050] In that regard, each quadrant U1 , U2, F1 , F2 of the validation slide 36, as shown in FIG. 6B, may include a suitable control, such as a peptide spot 1 14, cultured cells (not shown), a control tissue (not shown), or other as appropriate for evaluating antigen binding of the antibody (Block 1 16). Each spot 1 14 may include a peptide

(antibody, etc.) having the epitope of interest, (for example, the illustrative spots 1 14 may include a portion of the HER2 protein to which the polyclonal antibody included in the HERCEPTEST (Dako Denmark A/S, Glostrup, Denmark) binds for detecting breast cancer. The peptide may be prepared in a conventional manner and harvested from cell lines that are genetically engineered to over express the particular antigen.

[0051] The peptide spot(s) 1 14 may vary in volume, for example, ranging from microliters (such as is applied by inkjet printing or as used in microarrays) to milliliters, which may be dispensed by a pipette or other labware device. Furthermore, the peptide spot(s) 1 14 may be spatially arranged, as desired, on the slide 102 with one or more fiduciary markers such that the controller 74 may localize the position of the peptide spot(s) 1 14 on the capture image. With microliter droplets and fiduciary markers for localization, a plurality of peptide spots 1 14 may be used for simultaneously testing a plurality of parameters of the IHC procedure.

[0052] In accordance with one exemplary embodiment, one or more controls may be configured to assess antigen retrieval. As shown, the duplicate set of spots 1 14, e.g., those spots 1 14 within F1 and F2, may then be overspotted to control for sample pre-treatment, for example, with a casein spot 1 18 (Block 120). Casein is a phosphoprotein used for assessing antigen retrieval, as described in greater detail below. The overspot 1 18 may have a diameter that is generally larger than a diameter of the peptide spots 1 14.

[0053] The overspot 1 18 may, optionally, be further processed (Block 122). One such method of processing may include, for example, cross-linking the casein of the overspot 1 18 by immersing only the F1 and F2 quadrants into a formalin bath 124 while the U1 and U2 quadrants remain outside the formalin bath 124. The F1 and F2 quadrants remain within the formalin bath 124 for a sufficient period of time (for example, ranging from about 12 minutes to about 40 minutes at room temperature). For some antibodies, antigen retrieval is necessary to provide or enhance access of the antibody to the epitope or antigen. In other words, incomplete antigen retrieval reduces or hinders accessibility of the antibody to the epitope or antigen, and if the latter occurs, the interpretation would be a false negative result or a wrongly scored result. Once the cross-linkage is complete, the validation slide 36, with the now fixed spots 126, may be removed from the formalin bath 124, dried, and prepared for use with the sampled tissue 1 12.

[0054] In preparing a sample, and with reference now to FIGS. 7 and 8A, a tissue specimen (not shown), is acquired, for example, via biopsy or autopsy, undergoes formalin fixation (or other suitable fixative solution), is processed and embedded within paraffin, sliced by a microtome, mounted to the validation slide 36, and bake (Block 128) all in a manner that is generally known to those of ordinary skill in the art.

[0055] With reference now to FIG. 8B with FIG. 7, the validation slide 36 is pre- treated in accordance with the IHC protocol (Block 130). Consistent with the illustrative example, the pre-treatment may include antigen retrieval (Block 130). In that regard, the validation slide 36 may be immersed in a warmed buffer solution bath 132 for a period of time (for example, 40 minutes at 97 °C). Antigen retrieval is necessary for releasing the antigen from the fixative solution such that it may react with the antibodies of the IHC stains. In other words, incomplete antigen retrieval reduces the number of epitopes, if present in the tissue sample 1 12, available for reaction with IHC antigens and a false negative diagnosis may result.

[0056] In either case, the tissue sample 1 12 and the controls (i.e., the peptide spots 1 14) of the validity slide 36 are treated with the IHC stain in accordance with the staining protocol and, optionally, a cover slip (not shown) if no further processing is necessary (Block 134). Alternatively, if desired and/or necessary for the particular IHC protocol, a counter-stain may be applied prior to the cover slip (Block 136). For example, the validation slide 36 may be counter-stained with haematoxylin, which reacts with fixed spot 126, if present, and has a blue color.

[0057] If the peptide (i.e., antigen) is present and released, then the antibody, may react with the antigen and develop as a now stained tissue 138. The

antibody/stain also stains the peptide spots 1 14 of the staining control groups U1 , U2 and any peptide within the spots 1 14 of the pre-treatment control groups F1 , F2 uncovered by the removal of the overspotting. More particularly, in FIG. 8C, the peptide of the staining control groups U1 , U2 acquire the antibody stain ("stained peptide spots" 140) because the epitope within these quadrants was not previously hidden within the fixed spot 126 and is available for receiving the stain. Likewise, the peptide spotted in the pre-treatment control groups F1 , F2 acquire the antibody stain (also referred to as stained peptides spots 140) because the peptide, though previously hidden by the overspot 1 18, has been fully retrieved by removal of the overspot 1 18 and made available to receiving stain.

[0058] Conversely, and as shown in FIG. 8D, the peptide within the pre- treatment controls F1 , F2 is not, or is only partially, released from the overspot 1 18 and available for antibody staining (partial fixed peptides spots 126) (Block 130, FIG. 7). Because the antigen within the peptide present in the staining control groups U 1 , U2 was not previously hidden by an overspot 1 18, these spots acquire stain.

[0059] With the IHC protocol complete, and prior to pathological evaluation of the stained tissue 138, the validation slide 36 may be validated with the IHC device 20 (FIG. 1 ). In that regard, and with reference to FIG. 9, as well as FIGS. 1 -3, a method of validating the procedure performed on the validation slide 36 is described in

accordance with one embodiment of the present invention.

[0060] The user opens the drawer 28 of the IHC device 20, places the validation slide 36, with the stained tissue 138 thereon, into the validation slide cradle 32 of the slide tray 30, and closes the drawer 28 (Block 150). The validation slide 36, within the IHC device 20, is positioned between the light source 44 and the camera 46 and/or mirror 48 for acquiring an image of the validation slide, which may then be processed, if desired, for analysis (Block 151 ). The acquired image may then be used for evaluating and validating the performed pre-treatment and IHC protocol (Block 152). While the particular validation method, as described herein, is performed within the IHC device 20, it would be readily understood that such validation may be performed in accordance with other methods (such as visual inspection) with or without the IHC device 20.

[0061] The illustrative method of validating antigen retrieval and IHC staining is described with reference to FIGS. 1 -3 and 10. Imaging assembly 40 may be used to acquire an image of the loaded validation slide 36 and the image transferred to the controller 74 for image processing. A parameter of the stained peptide spots 140 of the pre-treatment control group F1 , F2 may be measured (Block 154), for example, the diameter of each spot may be determined by counting a number of pixels extending diametrically across the spot. If more than one control is used within the pre-treatment control group F1 , F2, then the parameters (or specifically, the diameters) of the stained peptide spots 140 may be evaluated separately or the mean (and/or median, and/or maximum) diameter of all stained spots 140 determined for evaluation. If the parameter is less than a predetermined range (for example, less than 0 and in which no diameter is detected indicating that the spot 140 is not present), then the IHC device 20 will determine whether any stained peptide spot 140 within the staining control groups U1 , U2 may be detected ("NO" branch of decision block 156). If no stained peptide spot 140 within the staining control group U1 , U2 is detected ("NO" branch of decision block 158), then an "Antibody Error" is returned (Block 160).

[0062] In some embodiments of the present invention, the IHC device 20 may further generate an indication of the type of antibody error returned. That is, by comparing the parameter of the stained peptide spots 140 of the pre-treatment control group F1 , F2 with the predetermined range in combination with evaluating the presence of the stained peptide spot 140, then one or more antibody errors may be returned. Such antibody errors may include, for example: (1 ) the antibody was omitted during the performed IHC protocol, (2) the incubation slide was incubated for an insufficient amount of time, or (3) the antibody reagent was over-diluted, for example, by a ratio of 1 :8, 1 :4, or 1 :2.

[0063] Returning again to block 158 of FIG. 10, if at least one stained peptide spot 140 within the staining control group U1 , U2 is detected ("YES" branch of decision block 158), then a "Reagent Dispensing Error" is returned (Block 162) and the process ends. The reagent dispensing error may include, for example, incomplete coverage of the validation slide 36 with IHC stain.

[0064] If measureable, then the stained peptide spots 140 are detected within the pre-treatment control group F1 , F2 (i.e. stained peptide spots 140 have a diameter greater than 0) ("YES" branch of decision block 156), then the parameter of each stained peptide spot 140 (or the mean or median of all spots) may be compared with the predetermined range (Block 166). In the illustrative example, because the overspot 1 18 (FIG. 6C) are larger than the peptide spots 1 14 (FIG. 6B), complete antigen retrieval may be confirmed by direct evaluation of the measured area of any remaining fixed spot 126. That is, pre-treatment (i.e., antigen retrieval) may be confirmed (Block 168) when the area of the peptide spots 1 14 of the pre-treatment control groups F1 , F2 falls within an predetermined range of areas (for example, ranging from about 2 mm² to about 5 mm²) ("YES" branch of decision block 166). Antigen retrieval failure ("NO" branch of decision block 166) is determined when the measured area of the fixed spots 126 of the pre-treatment control groups F1 , F2 are greater than the predetermined range, for example, greater than 8 mm². In that instance, a "Pre-Treatment Error" is returned (Block 170) and the process ends.

[0065] As was described above, the IHC device 20 may be further configured to determine a type of pre-treatment error that occurred during performance of the IHC protocol. Such errors may include, without limitation: (1 ) omitting a reagent, (2) insufficient retrieval time, or (3) insufficient temperature of the buffer bath.

[0066] With antigen retrieval evaluated, the device 20 may then evaluate the staining control groups U1 , U2. In that regard, a parameter (for example, the diameters) of the stained peptide spots 140 of the staining control groups U1 , U2 are measured (Block 172) and, if greater than 0 ("YES" branch of decision block 174), then performance of the IHC staining procedure is validated (Block 178). Otherwise, if no stained peptide spots 140 within the staining control groups U1 , U2 is detected, then an "Antibody Error" is returned (Block 176). The antibody errors may include those that were described previously or others as would be determinable by comparison of the parameter of the staining control groups U1 , U2 with the predetermined range.

[0067] Although not specifically shown, but in accordance with another embodiment of the present invention, completeness of the antigen retrieval may be determined from whether any of the casein spot 1 18 (FIG. 6C) remains. That is, because casein, with its haematoxylin counter-stain, has a characteristic blue color, the detection of any blue color (an increased signal in a blue channel of an RGB image, i.e. decreased optical density) within the fixed spot 126 would indicate that casein- haematoxylin complex remains and the antigen retrieval was incomplete.

[0068] Returning to FIG. 9, if the antigen retrieval and IHC protocol pass validation ("PASS" branch of decision block 180), then the process continues to validate the staining procedure (Block 182); otherwise ("FAIL" branch of decision block 180), the process ends.

[0069] Validating the staining procedure is described in greater detail with reference to FIG. 1 1 and with continued reference to FIGS. 1 -3. Specifically, the imaging assembly 40 may be used to acquire an image of the loaded validation slide 36 (Block 186), if an image was not previously acquired. To this end, camera 46 may include an imaging sensor, such as a Charge Coupled Device ("CCD") or other suitable sensor, having a plurality of light sensitive devices in a planar arrangement. Each light sensitive device comprising the imaging sensor typically produces a voltage

proportional to the amount of light incident on the device. The imaging sensor may thereby generate an electrical representation of an image comprised of a matrix of voltages, with each voltage representing a picture element (or pixel) having an optical density proportional to the representative voltage. Each voltage is, in turn, converted to a digital value representative of the imaging device voltage level and having an address corresponding to the location of the device within the imaging sensor. The camera 46 thereby produces a digital image file comprising a matrix of digital numbers that may be transferred to the controller 74 for image processing.

[0070] Each pixel comprising the digital image may have a spectral property related to the color of the portion of the digital image represented by the pixel. This spectral property, such as color or optical density, is typically represented by a plurality of color components, or channels, with each color channel having an intensity, or density, related to the color of the pixel. Color digital images typically classify pixels by determining red, green, and blue color channel densities for each pixel, although other color components, such as cyan, magenta, yellow, and black may also be used. Each pixel in the digital color image may therefore be represented by three color channel optical densities, with each color channel optical density represented by a digital value. By way of example, one common digital color imaging format uses three 8 bit values (one each for the red, green and blue color channel optical densities) to generate a 24 bit binary number that represents the color and brightness of each pixel of an image. To provide color images, the imaging sensor will therefore typically include light sensitive devices that are sensitive to different color channels. To this end, devices that are sensitive to red, green, and blue light may be arranged in a pattern on the imaging device. Each pixel in the resulting image may thereby be provided with red, green, and blue color channels, with each channel represented by a digital value proportional to the optical density of the component color of the image region represented by the pixel.

[0071 ] Thus, in a preferred embodiment, the camera 46 includes red, green, and blue color channels that provide three signals (one for each color) for each pixel of the acquired image. Thus, a tissue stained with a brown stain chromagen (such as the diaminobenzidine ("DAB") stain used in evaluating the presence of the HER2 protein) transmits red and green wavelengths of light while absorbing blue wavelengths of light (i.e. increasing optical density in the blue channel). Therefore, while the signal of any (or all) channel(s) may be considered in determining staining validity, use of the blue channel may be beneficial as the signal is used in detecting the DAB stain.

Accordingly, signal acquisition may be expedited and memory resources conserved, if desired. One of ordinary skill in the art will readily appreciate that the determination of the appropriate color channel signal to consider is directly related to the color generating results of the stains used in a particular IHC protocol.

[0072] In another embodiment of the present invention, rather than using the signal of one or more color channels, the perceived color (such as is measured by transmission UV-Vis spectroscopy) of each pixel may be evaluated and the number of such pixels counted. For example, the number of red pixels (illustrated as "PR"), blue pixels (illustrated as "PB"), and green pixels (illustrated as "PG") within each stained peptide spot 140 (FIG. 8C) within the staining control groups U1 , U2 may counted (Block 188).

[0073] If the color channel signal (or the number of PR, PB, and PG) fall within a predetermined range, as determined by a standard operating procedure training method described in detail below ("YES" branch of decision block 190), then the IHC device 20 returns an indication that the staining procedure has been validated (Block 192). Otherwise, if the color channel signal (or one or more of PR, PB, and PG) fall outside of the predetermined range ("NO" branch of decision block 190), then an "Antibody Error" occurred during the staining procedure and the IHC device 20 returns an error (Block 194) and the process ends.

[0074] In practice, the IHC device 20 may consider the stained peptide spots 140 of each staining control group U1 , U2 individually or, if performed in multiplicity, the mean, median, or maximum signal(s) or number of pixels for all stained peptide spots 140 of the staining control groups U1 , U2 may be used.

[0075] In any event, the IHC device 20 may display a result of the validation (Block 196; FIG. 9) such that the user may open the drawer to remove the validation slide 36 and, for example, transfer the slide 36 to the pathology department (if validated) for tissue diagnosis or dispose of the slide 36 in the proper manner (if invalidated) (Block 198).

[0076] In still other embodiments, the staining control groups U1 , U2 may include a plurality of control samples (spots, cells, tissues, etc) that vary in the concentration and/or presence of the antigen. For example, the staining control groups U1 , U2 may include a known 1 + grade tissue, a known 2+ grade tissue, and a known 3+ grade tissue. Accordingly, the 1 + grade tissue will have less antigen than the 3+ grade tissue and will stain less (have less color) than the 3+ grade tissue. Thus, the color channels signal (or pixel number) for each control sample of the staining control groups U1 , U2 will have an associated predetermined range for indicating staining validity. If one or more signals of the control samples of the staining control groups U1 , U2 are outside of the respective predetermined range, then an "Antibody Error" may be returned and the validation slide 36 rejected.

[0077] FIGS. 12A and 12B are exemplary screen captures 200, 202 for a validated slide and an invalidated slide, respectively. In each screen capture 200, 202, a screen identifier 204 provides an indication of what information is specifically shown

(illustrated as "Inspection Results"), and a time section 206 provides the date and time at which the validation process was completed. A results section 208 provides details as to whether the particular validation slide 36 under evaluation has "PASS" or "FAIL" the various validations. The results section 208 may also include the details of the specific results for the validation slide 36 under evaluation, with or without also providing the predetermined ranges for each detail. One or more arrows 210, 212 allow the user to scroll the results section 208 if information is hidden from view. Also, one or more navigational buttons 214, 216 (illustrated as "Back" and "Ok") may be used to move through various screens, and a help button 218 may be used to obtain additional information, if necessary or desired.

[0078] With reference now to FIG. 13, as well as further reference to FIGS. 1 -3 and 8A-8D, a training method according to one embodiment of the present invention is shown and described in greater detail. Because various laboratories (a laboratory technicians within the same laboratory) may perform slight variations to a particular IHC protocol, whether intentional or unintentional, the IHC device 20 may be trained to identify the predetermined range of results that are acceptable for the particular laboratory in which the device 20 resides. Exemplary variations may be due, in part, to the temperature of the lab, the particular equipment used in preparing reagents, the equipment (such as the temperature of the incubators) used in staining the IHC slides, or a particular alteration or counter stain that is used in accordance with a pathologist's preferences.

[0079] To determine the predetermined range of results the user in the particular laboratory prepares a plurality of validation slides 36 (for example, five or more without tissue samples 1 12) in accordance with the staining protocol for each of a plurality of days (for example, five days) (Block 220). After all staining protocols are complete, all stained IHC slides 36 are evaluated by the IHC device 20 (Block 222). For example, the color channel signals (or PR, PB, and PG) for each control may be measured to determine optical densities for color channels corresponding to each of the stained peptide spots 140 on each of the validation slides 36. These parameters (e.g., signals pixel numbers, for example) may then be combined to determine one or more statistical values, or a distribution of acceptable values for validation slides 36 (Block 224). If desired, the distributions may be fit to a particular distribution curve (e.g., Gaussian, bi- model, etc.) and the acceptable deviations within each determined distribution and a predetermined range of values (or standard deviation) may be determined (Block 226). The predetermined range of values may be based on, for example, a mean and standard deviation calculated for an optical density associated with a color channel of the stained peptide spots 140 in a particular location or associated with a slide set from a particular laboratory. The predetermined ranges and deviations may be saved within the memory 84 of the controller 74, or other storage device for evaluating validation slides 36 as described above. [0080] If so desired, then the IHC device 20 may be further configured to determine whether an improper slide is being used for the training method. For example, if one or more controls are missing, then the IHC device 20 may reject the results of the improper slide and request an additional slide. Furthermore, if a particular slide provides results that are statistically significant as compared to the other prepared slides (for example, more than three standard deviations or greater than a specified percentage), then the IHC device 20 may automatically reject the results or query the user as to whether to reject the results.

[0081] When evaluating the validity of the staining protocol of the validation slides 36 via image acquisition and processing, it is necessary to maintain the components of the imaging assembly 40 in an operable condition. Particularly for image processing, the light source 44 and the camera 46 must be properly aligned, focused, and the image color balanced in order to acquire a representative image of the validation slide 36 for staining validation. Alignment, focus, and image color balance not only affect the spatial resolution of the image, but also the color channel signals. Therefore, the operational conditions of the imaging assembly 40 may be calibrated, such as the method according to embodiments of the present invention and described with reference to FIGS. 14 and such calibration may be validated as described with reference to FIG. 15.

[0082] Two calibration slides 38, 38' according to different embodiments of the present invention are shown in FIG. 16A and 16B and include one or more optical tests for evaluating the operational conditions of the imaging assembly 40. While four optical tests 230, 232, 234, 236 are specifically shown, any number of tests may be included. A first optical test 230, 230' includes a plurality of gray scale bands 232, differing in an intensity of gray. For example, the calibration slide 38 of FIG. 16A includes three gray scale bands 232 of 71.25%, 25.74%, and 4.68% gray intensity. Calibration slide 38' of FIG. 16B includes four gray scale bands 231 of 71 .25%, 35.34%, 12.41 %, and 2.35% gray intensity. The ability of the imaging assembly 40 to distinguish the gray scale bands 231 is directly related to the ability of the imaging assembly 40 to determine RGB pixels.

[0083] A second optical test 232 may include, for example, a plurality of black dots. The black dots may be configured for determining the image sharpness and the ability to distinguish signals in each of the RGB color channels.

[0084] A third optical test 234 may include, for example, one or more pluralities of lines, which may be non-uniform in thickness and separation. The plurality of lines may be configured for determining the imaging assembly's ability to resolve each line (i.e., image resolution), which may allow the system to determine if the imaging assembly is properly focused.

[0085] A fourth optical test 236 may include, for example, a two-dimensional bar code. The bar code may be used to identify the particular calibration slide 38, 38' loaded and for retrieving the specifications of each from the controller 74 (FIG. 5) or other storage device, such as the LIS server 78.

[0086] With reference to FIG. 14, for use in the calibration operation, the calibration slide 38 is loaded into the calibration slide cradle 34 of the slide tray 30. The calibration slide cradle 34 is positioned distal to the validation slide cradle 32 such that the calibration slide 38, when loaded, remains within the IHC device 20 even with the drawer 28 opened to load/unload an validation slide 36. The calibration operation may optionally include a white balance operation (Block 240) performed when the drawer is closed (Block 239) without a validation slide 36 in the validation slide cradle 32.

[0087] The IHC device 20 determines whether the drawer 34 is in the open position (Block 241 ). For example, the IHC device 20 may ask the user to open the drawer or confirm that the drawer is in the open position. As an alternative, the IHC device may include a sensor to detect the position of the drawer. If the drawer 34 is not open ("NO" branch of decision block 241 ), then the process returns and the device 20 awaits a signal indicative that the drawer 34 is open. When the drawer 34 is open ("YES" branch of decision block 241 ), then the calibration slide 38 within the calibration slide cradle 34 of the slide tray 30 is positioned between the light source 44 and the camera 46 and an image of the calibration slide 38 may be acquired (Block 242).

[0088] Optical data, related to one or more of the optical tests 230, 234, 236, 238, may be determined by the controller 74 (FIG. 5) from the acquired image (Block 244) and compared to stored predetermined optical data for the particular calibration slide 38 stored in the controller (Block 246). Stored predetermined optical data may include actual optical values for the calibration slide 38 determined with another device (or supplied by the manufacturer) or optical data from a previous calibration operation in the same IHC device 20.

[0089] The optical data may be determined from the entire area of the desired optical test 230, 234, 236, 238, or from a subset of areas of the desired optical test. For example, with reference to FIGS. 18A and 18B, the first optical test 230, 230' of calibration slide 38, 38' includes a plurality of gray scale bands 231 , 232, differing in an intensity of gray. The optical data may be determined from a subset of areas from each gray scale band that is representative of the entire the gray scale band. For example, optical data may be determined from three portions of a gray scale band such as near opposite edges of the band and an intermediate portion. Determining optical data from a subset of the acquired image requires less processing time than would be necessary to determine optical data from the entire optical test area. Similarly, comparing optical data from a subset of the acquired image with the actual optical values or optical values from a previous calibration operation also requires less processing time.

[0090] To validate the calibration operation utilized above, and with reference to FIG. 15, the IHC device 20 determines whether the drawer 34 has been opened for the user to load/unload a validation slide 36 (Block 260). For example, the IHC device 20 may ask the user to open the drawer or confirm that the drawer is in the open position. As an alternative, the IHC device may include a sensor to detect the position of the drawer. If the drawer 34 is not open ("NO" branch of decision block 260), then the process returns and the device 20 awaits a signal indicative that the drawer 34 is open. When the drawer 34 is open ("YES" branch of decision block 260), then the calibration slide 38 within the calibration slide cradle 34 of the slide tray 30 is positioned between the light source 44 and the camera 46 and an image of the calibration slide 38 may be acquired (Block 264).

[0091] Optical data, related to one or more of the optical tests 230, 234, 236, 238, may be determined, by the controller 74 (FIG. 5) from the acquired image (Block 264) and compared to stored predetermined optical data (Block 266). Stored predetermined optical data may include actual optical values for the calibration slide 38 as determined with another device or supplied by the manufacturer or optical data from a previous calibration operation. The optical data may be determined from the entire area of the desired optical test 230, 234, 236, 238, or from a subset of areas of the desired optical test as described above with reference to the calibration operation. If the determined optical data values are significantly different as compared with the stored optical data values ("FAIL" branch of decision block 268), then an error is returned (Block 270) and the process ends. Another calibration procedure will likely be necessary before further use of the imaging assembly 40. Otherwise, and if the determined optical data values are sufficiently close to the stored optical data values to indicate that the imaging assembly 40 is functioning properly ("PASS" branch of decision block 268), then the process may return to await another open drawer (Block 260). One will appreciate that the calibration operation and the validation operation may be conducted simultaneously or serially and need not be conducted as separated operations.

[0092] Errors in the image analysis of the optical tests 230, 234, 236, 238 may also provide information with respect to the optical path 49. For example, particulate, contaminants, or camera lens defects may be detected by calibration errors.

Additionally, if no image acquisition occurs, or if image acquisition occurs with improper optical parameters, then the calibration errors may indicate that the light source 44 and/or the camera 46 are inoperable.

EXAMPLE

[0093] The data provided in this example were collected with two test slides having control groups arranged similar to the control group arrangement shown if FIG. 8A. Namely, each test slide had four circular control groups arranged in two rows of two. The control peptide for each control group was a portion of the HER2 protein, which is the antigen for the polyclonal antibody provided with the HERCEPTEST kit used for detect cells the overexpressing the HER2 protein, which is indicative of breast cancer cells in a tissue sample. HER2 peptides were suspended in a solution and applied to the surface of the slide in the desired location. After application of HER2 peptide, the HER2 peptide spots were overspotted with casein. The casein overspot had a larger area than the area of the HER2 peptide spots. The casein overspot in row 1 of the control groups were cross-linked in a formalin bath using established procedures, whereas the casein overspots for the row 2 control groups were not cross- linked. After cross-linking, the test slide was processed using the established

HERCEPTEST procedure. Briefly, test slide 1 was immersed in an antigen retrieval solution for about 40 minutes at about 95 to 99 degrees C to release the antigen from the casein overspot and then washed. The antigen retrieval step was skipped for test slide 2. The test slides were exposed to a peroxidase-blocking reagent containing 3 percent hydrogen peroxide. After peroxidase blocking, the test slides were washed and exposed to the HER2 polyclonal antibody. The slides were then washed and exposed to a visualization reagent that included a dextran polymer conjugated with horseradish peroxidase and affinity-isolated goat anti-rabbt immunoglobulins. After rinsing, the test slides were exposed to a DAB solution, washed and visualized in a IHC device with an IHC protocol in accordance with embodiments of the invention.

[0094] Table 1 , below, includes data collected from the two test slides and includes intensity data for ODGry (combination RGB with a green bias), ODRed (red),

ODGrn (green) and ODBIu (blue). The data demonstrate that with DAB staining, blue wavelengths of light (ODBIu) have the greatest signal intensity. The mean intensity is the average for the two spots in a row for a particular color (i.e., the Mean ODRed value for row 1 is the average of the ODRed intensity values observed for the two control groups in that row).

[0095] In both slides, the ODBIu signal intensity is greater for the control groups from row 2 because the casein overspots in row 2 were not cross-linked. These data suggest that for at least DAB staining, observation of light in the blue spectrum is superior to light observed in other portions of the visible spectrum, as well as combination RGB light, because the greater ODBIu signal intensity gives better resolution over background signals.

[0096] In slide 2, the observed area of intensity is greater for the control groups in row 1 than they were for the control groups in row 2 because the antigen retrieval step was skipped when processing this slide, leaving the fixed casein overspot. These data indicate that the IHC device can detect an improperly processed slide (i.e., that the antigen retrieval step was skipped) by comparing the area of intensity between the control group in row 1 in which the overspot was not cross-linked with the area of intensity of the control group in group 2 in which the overspot was cross-linked.

[0097] While the present invention has been illustrated by description of various embodiments and while those embodiments have been described in considerable detail, it is not the intention of applicant to restrict or in any way limit the scope of the appended claims to such details. Additional advantages and modifications will readily appear to those skilled in the art. The invention in its broader aspects is therefore not limited to the specific details and illustrative examples shown and described. Accordingly, departures may be made from such details without departing from the spirit or scope of applicants' invention.

What is claimed is:

Claims

1 . A method of validating an immunohistochemical ("IHC") slide pre-treated and stained in accordance with an IHC protocol, the IHC slide including a validation portion having at least one control group thereon configured to validate at least one parameter directed to at least one of pre-treatment or of staining of the IHC slide, the method comprising:

capturing an image of the IHC slide;

measuring a value of the at least one parameter of the at least one control group based on the captured image;

comparing the measured value of at least one parameter to an acceptable value; and

based on the comparison, generating an indication of the validity of the

IHC slide.

2. The method of claim 1 , wherein the at least one control group includes a pre-treatment control group and a staining control group.

3. The method of claim 2, wherein the staining control group includes a first quantity of a peptide, the peptide having an antigen configured to react with a stain of the IHC protocol.

4. The method of claim 2, wherein the pre-treatment control group includes a first quantity of a peptide, the peptide having an antigen configured to react with a stain of the IHC protocol only after an antigen retrieval method.

5. The method of claim 1 , wherein the at least one control group includes a peptide spot and the at least one parameter is a spatial character of the peptide spot.

6. The method of claim 5, wherein the spatial character is one of a diameter or an area, the method further comprising:

in response to the comparison, wherein the diameter or area of the peptide spot is greater than an acceptable value, generating an indication of an error in the pre-treatment of the IHC slide.

7. The method of claim 1 , wherein the at least one parameter is an optical density of at least one color in the image of the at least one control group and the method further comprises:

in response to the comparison, wherein the optical density of the at least one color is less than, or greater than, an accepted optical density, generating an indication of an error in the staining of the IHC slide.

8. The method of claim 1 , wherein the at least one control group includes a plurality of tissue samples, each of the tissue samples having a pathological grade.

9. A method of validating an immunohistochemical ("IHC") slide pre-treated and stained in accordance with an IHC protocol, the IHC slide including a validation portion having a pre-treatment control group comprising at least one pre-treatment control configured to validate the slide pre-treatment and a staining control group comprising at least one staining control configured to validate the slide staining, the method comprising:

capturing an image of the IHC slide;

detecting at least one parameter of the pre-treatment control; comparing the at least one parameter of the pre-treatment control to an acceptable value; and

based on the comparison, generating an indication of the validity of the IHC slide pre-treatment.

10. The method of claim 9, wherein the pre-treatment control of the pre- treatment control group includes at least one peptide spot and the at least one parameter includes a spatial character of the peptide spot.

1 1. The method of claim 10, wherein the spatial character is at least one of a diameter or an area, the method further comprising:

in response to the comparison, wherein the diameter or the area of the peptide spot is greater than the acceptable value, generating an indication of an error in the pre-treatment of the IHC slide.

12. The method of claim 1 1 , wherein the error in the pre-treatment of the IHC slide includes at least one of an incomplete antigen retrieval, no antigen retrieval, an improper buffer, or an improper buffer temperature.

13. The method of claim 9, further comprising:

in response to the parameter, wherein the at least one parameter of the pre-treatment control is within the acceptable value, generating an indication of a valid pre-treatment;

detecting a first optical parameter of the staining control;

comparing the first optical parameter of the staining control to a first acceptable optical value; and

based on the comparison, generating an indication of the validity of the IHC slide staining.

14. The method of claim 13, wherein the first optical property is an optical density of at least one wavelength of light, the method further comprising:

in response to comparison, wherein the optical density is less than, or greater than, the first acceptable optical value, generating an indication of an error in the staining of the IHC slide.

15. The method of claim 14, wherein the error in the staining of the IHC slide includes at least one of an antibody error, a reagent dilution, an incubation time, or an incubation temperature.

16. The method of claim 13, wherein the at least one parameter of the pre- treatment control group is a second optical parameter of the pre-treatment control.

17. The method of claim 13, wherein validating the pre-treatment control includes:

comparing the second optical parameter to the acceptable value, wherein the second optical parameter being within the acceptable value.

18. A method of validating an immunohistochemical ("IHC") slide that includes a validation portion with a fixed spot and an unfixed spot and that has been subjected to an IHC protocol that includes an antigen retrieval protocol and a staining protocol, the method comprising:

capturing an image of the IHC slide;

determining at least one parameter of at least one of the fixed spot and the unfixed spot based on the captured image; and

validating the IHC protocol based at least in part on the at least one parameter of the at least one of the fixed spot and unfixed spot.

19. The method of claim 18, wherein the at least one dimension includes a diameter of the fixed spot and validating the IHC protocol includes:

in response to the diameter of the fixed spot being less than or equal to a first value, generating an indication of an error in the pre-treatment of the IHC slide.

20. The method of claim 18, wherein the at least one dimension includes a diameter of the unfixed spot and returning an error includes:

in response to the diameter of the unfixed spot being greater than a second value, generating an indication of an error in the dispensing of a reagent.

21. The method of claim 20, wherein the first and second values are zero.

22. The method of claim 18, wherein the at least one dimension includes a diameter of the fixed spot and validating the IHC protocol further comprises:

in response to the diameter of the fixed spot being greater than a second value, generating an indication of an error in the antigen retrieval protocol.

23. The method of claim 18, wherein validating the IHC protocol includes:

determining an optical density level of at least one wavelength of light of the fixed spot; and

in response to the optical density level being higher than a first threshold, generating an indication of an error in the retrieval of the antigen.

24. The method of claim 23, wherein the optical density level is determined by a number of pixels in an area of the captured image corresponding to the fixed spot.

25. The method of claim 23, wherein the wavelength of light corresponds to one of red, green, or blue.

26. The method of claim 18, wherein validating the IHC protocol includes:

determining an area of the fixed spot; and

in response to the area of the fixed spot being outside a predetermined range, generating an indication of an error in the antigen retrieval protocol.

27. The method of claim 26, wherein the predetermined range is between about 2 square millimeters and about 5 square millimeters.

28. The method of claim 26, further comprising:

in response to the area of the fixed spot being within the predetermined range, determining a diameter of the unfixed spot; and

in response to the diameter of the unfixed spot being greater than a first value, generating an indication of validity of the staining protocol.

29. The method of claim 28, further comprising:

in response to the diameter of the unfixed spot being less than or equal to the first value, generating an indication of an error in the staining protocol.

30. The method of claim 18, wherein the at least one dimension includes a diameter of the unfixed spot and the method further comprises:

in response to the diameter of the unfixed spot being less than or equal to a predetermined value, generating an indication of an error in the staining protocol.

31. The method of claim 30, wherein the predetermined value is zero.

32. The method of claim 18, wherein validating the IHC protocol further comprises:

determining an area of the fixed spot;

in response to the area of the fixed spot being within a predetermined range, determining a diameter of the unfixed spot; and

in response to the diameter of the unfixed spot being greater than a predetermined value, generating an indication of the validity of the IHC slide.

33. The method of claim 32, further comprising:

in response to the area of the fixed spot being outside the predetermined range, generating an indication of an error in the antigen retrieval protocol.

34. The method of claim 32, further comprising:

in response to the diameter of the unfixed spot being less than or equal to the predetermined value, generating an indication of an error in the staining protocol.

35. The method of claim 32, wherein the predetermined range is from about 2 square millimeters to about 5 square millimeters and the predetermined value is zero.

36. The method of claim 18, wherein validating the IHC protocol further comprises:

in response to a color content of the unfixed spot, generating an indication of the validity of the staining protocol.

37. The method of claim 36, wherein validating the staining process includes: determining a number of pixels of an area of the captured image corresponding to the unfixed spot;

comparing the number of pixels to a predetermined number of pixels; and in response to the number of pixels being outside the predetermined number of pixels, generating an indication of an error in the staining protocol.

38. The method of claim 37, wherein the first color is one of red, green, or blue.

39. The method of claim 36, wherein validating the staining protocol includes:

determining a first number of pixels of an area of the captured image having a first color and corresponding to the unfixed spot;

comparing the first number of pixels to a first predetermined range;

determining a second number of pixels of the area and having a second color;

comparing the second number of pixels to a second predetermined range; determining a third number of pixels of the area and having a third color; comparing the third number of pixels to a third predetermined range; and in response to at least one of the first number of pixels being outside the first predetermined range, the second number of pixels being outside the second predetermined range, or the third number of pixels being outside the third

predetermined range, generating an indication of an error in the staining protocol.

40. The method of claim 39, wherein the first color is red, the second color is green, and the third color is blue.

41. A method of validating an immunohistochemical ("IHC") slide that includes a validation portion with a plurality of fixed spots and a plurality of unfixed spots and that has been subjected to a IHC protocol that includes an antigen retrieval protocol and a staining protocol, the method comprising:

determining a spectral property of at least one spot of the plurality of fixed spots and at least one of the plurality of unfixed spots; and validating the IHC protocol based at least in part on the spectral property of the at least one spot of the plurality of fixed spots and the at least one of the plurality of unfixed spots.

42. The method of claim 41 , wherein determining the spectral property of the at least one spot of the plurality of fixed spots and the at least one of the plurality of unfixed spots further comprises:

determining an optical density level for at least one color channel in a first area corresponding to the at least one spot of the plurality of fixed spots and in a second area corresponding to the at least one spot of the plurality of unfixed spots.

43. The method of claim 41 , wherein determining the spectral densities further comprises:

determining a first optical density level for at least one color channel in a first area corresponding to a first one of the plurality of fixed spots and a second area corresponding to a first one of the plurality of unfixed spots;

determining a second optical density level for the at least one color channel in a third area corresponding to a second one of the plurality of fixed spots and a fourth area corresponding to a second one of the plurality of unfixed spots; and

calculating a mean value of the first and second optical density values for each of the plurality of fixed spots and the plurality of unfixed spots.

44. The method of claim 41 , further comprising:

capturing an image of the IHC slide;

determining the optical density level for at least one color channel of a first area of the captured image corresponding to at least one of the plurality of fixed spots and of a second area of the captured image corresponding to at least one of the plurality of unfixed spots includes; and

determining a number of pixels associated with the first and second areas.

45. The method of claim 41 , wherein the at least one color channel is a blue color channel and validating the IHC protocol based at least in part on the spectral property of a first one of the plurality of fixed spots further comprises:

generating an indication of an error in the antigen retrieval protocol in response to the optical density level of the blue color channel of the first one of the plurality of fixed spots being greater than a first threshold.

46. A method of training an apparatus for validating an immunohistochemical ("IHC") slide, the method comprising:

preparing and staining a plurality of reference slides in accordance with an IHC protocol, each of the plurality of reference slides including a validation portion having at least one control;

capturing an image for each of the plurality of reference slides; identifying an area of each of the captured images corresponding to the at least one control of a respective one of the plurality of reference slides;

determining a characteristic of the at least one control from the each of the captured images; and

calculating a first statistical value based on the characteristic determined from each of the captured images and corresponding to the respective ones of the plurality of reference slides.

47. The method of claim 46, further comprising:

saving the statistical value for comparing with an IHC slide having a tissue sample thereon and pre-treated in accordance with the IHC protocol.

48. The method of claim 46, wherein identifying the at least one control includes identifying a spatial coordinate.

49. The method of claim 46, wherein identifying the at least one control includes identifying an optical property.

50. The method of claim 46, wherein calculating the first statistical value is further includes determining the characteristic of each of a plurality of controls within the validation portion of each of the plurality of reference slides, wherein each of the plurality of controls has a position within the validation portion.

51. The method of claim 46 wherein the characteristic type is a color of the at least one control.

52. The method of claim 46, wherein the characteristic is a spatial dimension of the at least one control.

53. The method of claim 46, wherein preparing the plurality of reference slides includes:

treating each of the plurality of reference slides with an antigen retrieval protocol to release at least one antigen within the at least one control; and

treating each of the plurality reference slides with a staining protocol having at least one antibody configured to react with the at least one released antigen.

54. The method of claim 53, wherein the antigen retrieval protocol includes removing a fixing agent.

55. The method of claim 46, wherein at least a portion of the plurality of references slides are pre-treated and stained on each of a plurality of days.

56. The method of claim 46, wherein determining the characteristic further comprises:

determining a number of pixels having a first color within the area of each of the captured images.

57. The method of claim 46, wherein the statistical value is at least one of a mean value, a median value, a standard deviation, a minimum value, a maximum value, or a mode.

58. The method of claim 46, wherein validating the IHC slide based on the statistical value further comprises:

capturing an image for the IHC slide;

determining the characteristic for at least one control of the IHC slide from the captured image of the IHC slide;

comparing the first statistical value to the characteristic determined from the captured image of the IHC slide.

59. The method of claim 58, wherein the characteristic of the at least one control of the IHC slide is a number of pixels having a first color and that are in an area of the captured image of the IHC slide corresponding to the at least one control of the IHC slide, and the statistical value defines a predetermined range of values for the number of pixels having the first color.

60. The method of claim 46, wherein the characteristic is a first characteristic, the method further comprising:

determining a second characteristic of the at least one control from each of the captured images; and

calculating a second statistical value based on the second characteristic determined from each of the captured images and corresponding to the respective ones of the plurality of reference slides.

61. The method of claim 46, further comprising:

comparing the characteristic of the at least one control from each of the captured images to a predetermined characteristic limit; and

in response to the characteristic corresponding to a select one of the captured images being outside the predetermined characteristic limit, excluding the characteristic corresponding to the select one of the captured images from the first statistical value calculation.

62. The method of claim 61 , wherein the predetermined characteristic limit is at least one of a circularity limit, an area limit, or an intra-spot pixel variation limit.

63. The method of claim 62, wherein the predetermined characteristic limit is the circularity limit and is generated in response to a variance in a plurality of distance measurements extending from a point within the area corresponding to the at least one control of the select one of the captured images to a plurality of points on a boundary of the area exceeding the predetermined variance in the plurality of distance

measurements.

64. The method of claim 62, wherein the predetermined characteristic limit is the area limit and is generated in response to a variance in a number of pixels within the area corresponding to the at least one control of the select one of the captured images exceeding the predetermined variance in the number of pixels.

65. The method of claim 64, wherein predetermined characteristic limit is the intra-spot pixel variation limit and is generated in response to a variance in a number of pixels within the area corresponding to the at least one control of the select one of the captured images and having a color exceeding the predetermined variance in the number of pixels having the color.

66. The method of claim 65, wherein the predetermined characteristic limit is a predetermined range of pixel numbers.

67. An immunohistochemical ("IHC") validation device comprising:

an imaging assembly comprising a light source and a light receiving device;

an optical path operably coupling the light source to the light receiving device;

a slide tray including an IHC slide cradle and a calibration slide cradle, the slide tray being configured to be placed in a first position such that the IHC slide cradle is positioned in the optical path and in a second position such that the calibration slide cradle is positioned in the optical path.

68. The device of claim 67, wherein the imaging assembly further comprises a mirror positioned within the optical path and the light receiving device is vertically offset from the light source.

69. The device of claim 67, further comprising:

an optically transparent debris tray positioned between the slide tray and at least one of the light source and light receiving device.

70. The device of claim 67, wherein the light receiving device is a camera.

71. The device of claim 67, further comprising: a calibration slide positioned within the calibration slide cradle and having at least one optical test thereon.

72. The device of claim 71 , wherein the at least one optical test includes at least one of a grayscale range, a line pair, a fiducial marker, and a bar code.

73. The device of claim 71 , further comprising:

a processor; and

a memory operatively coupled to the processor and including instructions that, when executed by the processor, cause the processor to:

capture an image of the calibration slide;

measure a value of the at least one optical test;

compare the measured value of at least one parameter to a predetermined optical result; and

based on the comparison, generating an indication of an error in the device calibration.

74. The device of claim 67, further comprising:

a processor; and

capture an image of the IHC slide;

measure a value of the at least one parameter of the at least one control group;

compare the measured value at least one parameter to an acceptable value; and

based on the comparison, generating an indication of the validity of the

IHC slide.