WO2006037332A1

WO2006037332A1 - A system and method for biological sample processing comprising means for vibrating the sample during processing

Info

Publication number: WO2006037332A1
Application number: PCT/DK2005/000637
Authority: WO
Inventors: Lars Winther
Original assignee: Dako Denmark A/S
Priority date: 2004-10-06
Filing date: 2005-10-05
Publication date: 2006-04-13
Also published as: JP2008516203A; EP1797410A1; US20060073074A1; AU2005291666A1

Abstract

An automated sample processing system and methods are disclosed where sample(s) are arranged on a carrier element and a process operation control system automatically processes the sample(s) perhaps robotically according to protocol and according to a scheduling system. The processing may include and be enhanced by a vibration of the sample via a vibrator disposed in or upon a sample carrier or a sample cover. Alteration of an initial aggregated event topology may be accepted while the system is processing an initial aggregation and varied-parameter robotic control simulation functionalities may be accomplished to determine an enhanced sequence for processing.

Description

A SYSTEM AND METHOD FOR BIOLOGICAL SAMPLE PROCESSING COMPRISING MEANS FOR VIBRATING THE SAMPLE DURING PROCESSING

CROSS REFERENCES TO RELATED APPLICATIONS

This application claims the benefit of the filing date of and the right of priority to United States Provisional Application 60/616,444, filed October 6, 2004 and titled "Enhanced Sample Processing System and Methods of Biological Slide Processing", said Provisional application being incorporated by reference herein in its entirety. This application is also related to the following international applications and their respective United States National Stage Applications: international application PCT/DK2003/000877, having an international filing date of December 15, 2003; international application PCT/DK2003/000911, having an international filing date of December 19, 2003; international application PCT/US2003/040518, having an international filing date of

December 19, 2003; international application PCT/US2003/040880, having an international filing date of December 22, 2003; international application PCT/US2003/040591, having an international filing date of December 19, 2003; international application PCT/US2003/040520, having an international filing date of December 19, 2003; international application PCT/US20O5/006383, having an international filing date of February 28, 2005; international application PCT/US2003/041022, having an international filing date of December 22, 2003; international application PCT/US2003/040974, having an international filing date of December 19, 2003; international application PCT/US2003/040519, having an international filing date of December 19, 2003. This application is further related to co- pending United States Patent Application 10/741 ,628 filed on December 19, 2003 ; to co- pending United States Patent Application 10/731,316 filed on December 8, 2003; to co- pending United States Patent Application 11/119,417, filed on April 30, 2005 and to a co- pending United States Patent Application titled "Method and Apparatus for Automated Pre- Treatment and Processing of Biological Samples", filed on June 20, 2005 and having attorney docket number Pl 15US02. FIELD OF THE INVENTION

This application relates to the field of sample processing systems and methods of scheduling an aggregate of events for the processing of samples or the process system. The present invention may be directed to the automated processing, treatment, or staining of samples arranged on carriers, such as slides, and in some embodiments, directed to the continuous or batch processing of samples and carriers. Embodiments may further relate to control systems for sample processing and data input, acquisition, maintenance, and retrieval for sample processing. Applications to which the present invention may especially relate include immunohistochemistry, in-situ hybridization, fluorescent in-situ hybridization, special staining, and cytology, as well as potentially other chemical and biological applications.

BACKGROUND OF THE INVENTION

Sample processing in immunohistochemical (IHC) applications and in other chemical and biological analyses may require one or a number of various processing sequences or protocols as part of an analysis of one or more samples. The sample processing sequences or protocols may be defined by the individual or organization requesting an analysis, such as a pathologist or histologist of a hospital, and xnay be further defined by the dictates of a particular analysis to be performed. The protocol for the processing of a specific sample - as used herein - means a sequence of events such as processes that defines the processing of a specific sample in the automated sample processing system.

In preparation for sample analysis, a biological sample may be acquired by known sample acquisition techniques and may comprise, for example in IHC applications, tissues generally or even in some applications one or a plurality of isolated cells, such as in micro- array samples, and may be presented on a sample carrier including but not limited to microscope slides. Furthermore, the sample may be presented on the carrier variously and potentially in some form of preservation. A.s one example, a sample such as a layer or slice of skin may be preserved in formaldehyde and presented on a carrier with one or more paraffin or other chemical layers infiltrating the sample. Inimunologic applications, for example, may require processing sequences or protocols that comprise steps such as de-paraffinization, target retrieval, reagent application, and staining, especially for in-situ hybridization (ISH) techniques. In some applications, these steps may have been performed manually, potentially creating a time-intensive protocol and necessitating personnel to be actively involved in the sample processing. Even when performed automatically, there have been inefficiencies in such systems. Attempts have been made to automate sample processing to address the need for expedient sample processing and a less manually burdensome operation. However, such previous efforts may have not fully addressed certain specific needs for an automated sample processing system. Previous efforts to automate sample processing may be deficient in several aspects that prevent more robust automated sample processing, such as: the lack of sufficient computer control and monitoring of sample processing; the lack of information sharing for processing protocol and processing status, especially for individual samples; the lack of practical information input and process definition entry capabilities; the lack of diagnostic capabilities; and. the lack of real-time or adaptive capabilities for multiple sample batch processing.

Past efforts at automated sample processing for samples presented on carriers such as slides, such as US Patent No. 6352861 to Ventana Medical Systems, Inc. and LJS Patent No. 5839091 to Lab Vision Corporation, have not afforded the various advantages and other combinations of features as presented herein.

One of the various aspects that maybe significant to users of automated, process systems is that of allowing changes to the processing while it is ongoing. In this regard, it has often been considered that operators have to allow existing sequences to finish before inserting or changing the aggregate in some manner. In addition, operators often have needed particular knowledge and skills in order to assure the integrity of the process or instrument or result. The present invention seeks to reduce such effects to some degree and seeks to provide a system that may be considered more user, operator, supplier, or manufacturer friendly and may be adaptable to real-world conditions and events.

SUMMARY OF THE INVENTION The present invention presents an automated sample processing system that may greatly improve operation of automated sample processing from several perspectives. According to the invention, there is provided an automated sample processing system comprising a plurality of positions, each position arranged for receiving a sample carrier (e.g., a microscope slide) bearing at least one biologic sample, wherein the system may further comprise at least one vibrator means arranged to be adapted to get in toucli with a sample carrier or cover in order to transfer vibrations to a sample and/or reagents applied to the sample and wherein an actuation of the vibrator means is controlled by a protocol controlling the processing of the sample.

Preferably, each of the plurality of positions for sample carriers comprises at least one vibrator element arranged to vibrate a sample carrier or cover. In some embodiments only a fraction, such as 1/3, of the plurality of positions for sample carriers comprises at least one vibrator element arranged to vibrate a sample carrier or cover. In this context "plu rality" means any number exceeding 7, such as 10, 16, 20, 32, 40, 50 or 64, or even higher.

Preferably, the processing of the plurality of slides is scheduled by a multiple event scheduling program scheduling all processing steps specified in all protocols for samples present in the sample processing system. Such scheduling is disclosed in international patent application publication WO 2004/058404 A2, incorporated by reference herein in its entirety.

The vibrations applied to the sample and/or reagents and/or probes may Lmprove or speed-up mixing and reactions, and decrease reaction time under various processing steps, thereby enabling a faster processing of the sample. An optimal scheduling is essential in an automated processing system order to benefit from the time reductions made possible by the use of vibrations.

Preferably, the type of vibration, such as amplitude and frequency, continuous or pulsed, is selected in regards to or specifically adapted to each processing step. Where the reagent has a very high viscosity, the vibration may influence the properties of tlie reagent in such a manner that the viscosity decreases during the vibrations. The reagent may then spread, in favorable manner, over the sample and contact all areas of the sample. The vibrations may be acoustic waves. In preferred embodiments, the wavelength of the applied acoustic waves are smaller than or of the same order of size as the size of the sample.

An essential feature of the present invention is that the element providing the vibrations to a specific sample should be under precise and accurate control from the specific protocol controlling the sample processing of the specific sample. Even though small vibrations improve the processing during some processing steps, such vibrations may cause deterioration in other steps, and must be avoided in those steps. As an example, it could deteriorate the sample staining if such vibrations were applied during a chromogen treatment step.

The primary advantage of introducing vibrations of the sample and reagents or probes during a processing step is that the processing time needed to accomplish a specific processing step may be reduced considerably for a number of processing steps. Reducing processing time is a highly appreciated advantage. More specifically, the method is very advantageous for wash, deparaffinization, and re-hydration steps. For these steps, a processing time of e.g. about 5-10 min. may be reduced to about 1-3 minutes. For the antibody and visualization steps, the vibration appears to improve the process, providing a better signal. By using vibrations during a hybridization step, this step may be shorter, and the undesired drying will diminish.

In a preferred embodiment of an automated sample processing system in accordance with the invention, the at least one vibrator element is embedded in an element arranged to become in supporting contact with a sample carrier. Alternatively, in another preferred embodiment, the at least one vibrator element may be embedded in an element arranged to cover a sample on a sample carrier.

The automated sample processing system in accordance with the present invention may be of a kind wherein the sample carriers may be loaded on a carousel. Alternatively, in a different embodiment of an automated sample processing system, the sample carriers may be loaded continuously into an open drawer among a plurality of normally closed drawers, wherein the processing may continue in all closed drawers and wherein the processing may be stopped in a specified drawer in order to open the drawer for inserting sample carriers in the open drawer..

In a further embodiment of an automated sample processing system according to the present invention, sample carriers may be arranged in a plurality of separate locations, and moving means may be arranged to execute movement of a sample carrier from one location to another, and at least one of the locations may be provided for a specific processing step and at least one of the locations may comprise a vibrator element. In one preferred embodiment of an automated sample processing system according to the present invention, the plurality of separate positions is arranged in a compact rotary symmetric unit comprising several levels (floors) of positions enclosing or neighboring a transport means that is able to elevate and insert by translational movement a sample carrier to a specified level. Preferably the processing of the plurality of slides is scheduled by a scheduling program scheduling all processing steps specified in all protocols for samples present in the sample processing system.

In further embodiments of an automated sample processing system according to the present invention, the scheduling process is repeated after insertion of a new set of carriers with new samples, each having a specified protocol.

In preferred embodiments of an automated sample processing system according to the present invention, the processing comprises the application of vibrations during at least one processing step from the group consisting of the following processing steps: washing, de- paraffmization, re-hydration, target retrieval, sample conditioning, enzyme/antibody application, probe/reagent application, rinsing, de-hydration, drying and mounting. Preferably, the processing within an automated sample processing system according to the present invention comprises processes belonging to the group consisting of: in-situ hybridization (ISH), immunohistochemistry (IHC) techniques, fluorescent in-situ hybridization (FISH); special staining (SS) of samples, and micro-arrays; especially techniques incorporating target retrieval or the staining of samples.

In a preferred embodiment of an automated sample processing system according to the present invention, the control of the at least one vibrator element incorporates controlling the start and stop of the application of the vibrator element. Preferably, the control of the at least one vibrator element incorporates controlling the frequency of the vibrations. In such a situation, the optimal choice of frequency will depend on material properties. Preferably, the control of the at least one vibrator element incorporates controlling the pulse length or duration of an applied vibration. Preferably, the control of the at least one vibrator element incorporates sweeping the frequency of the vibrations. Preferably, the control of the at least one vibrator element incorporates modulating the frequency of the vibrations. Preferably, the control of the at least one vibrator element incorporates controlling the amplitude of the vibrations. Preferably, the control of amplitude may include an upper limit of the amplitude. Preferably, the upper limit of the amplitude may be adjusted.

In some preferred embodiments of an automated sample processing system according to the present invention, the scheduling and control of a vibrator element is related to the protocol defining the processing of the sample to be vibrated by the vibrator element, and the scheduling and control may change when a new sample, processed according to a different protocol, is loaded into a position to be vibrated by a vibrator element.

In some preferred embodiments of an automated sample processing system according to the present invention, the control of the at least one vibrator element incorporates, comprises or relates to an energy management system ensuring that no or only very little energy is distributed to the vibrator when no vibrations should be applied to the sample according to the specific protocol controlling the processing of the specific sample. In some preferred embodiments, the vibrator is activated for short period of between 1 and 10 seconds, preferably between 3 and 5 seconds and, then deactivated for a longer period. Preferably, the deactivation period is between 50 and 70 seconds. In an especially preferred embodiment of an automated sample processing system according to the present invention, the system is a stainer comprising a plurality of elements, each element arranged to support or be in touch with a microscope slide, wherein each element comprises a vibrator having the capability to transfer vibrations to a microscope slide, the stainer further comprising a plurality of vibration controllers that regulate electrical power to said vibrators, each said vibration controller being controlled in dependence of a specific protocol designed for controlling the processing of the sample on the slide.

In a preferred embodiment of a vibrator element for an automated sample processing system according to the present invention, the vibrator element is embedded in the carrier (e.g., microscope slide). Alternatively, the vibrator element maybe embedded in a support for the carrier. Preferably, the support also comprises heating means for increasing the temperature of the sample. Thus, the vibrator element may be embedded in a heating plate for increasing the temperature of the sample.

In a further embodiment of a vibrator element for an automated sample processing system according to the present invention, the vibrator element is embedded in a cover arranged to cover a sample on the carrier. Preferably, the vibrator element is arranged to contact the carrier, either in direct contact but preferably through a thin layer of a liquid. The vibrator element may be arranged to contact an edge of the carrier, or, alternatively, a surface of the carrier.

In a one embodiment of a vibrator element for an automated sample processing system according to the present invention, the vibrations are generated by a tapping robotic arm. In another embodiment, the vibrations are generated by an electric vibrator.

The vibrator element may be a piezo electric element and may be a laser actuated element.

In a presently preferred ambodiment a vibrator element for an automated sample processing in accordance with the present invention is a SAW chip, that generates surface acoustic waves (SAW). A method of automated sample processing in accordance with a preferred embodiment of the invention comprises the steps of: establishing an automated sample processing system having an automated process operation capability that causes automated process operation events through robotic sample process functions; loading a plurality carriers with biologic samples in the automated sample processing system; loading or accessing data enabling the sample processing system to define at least one protocol for the control of the sample processing of each of the loaded samples; and performing the sample processing using at least one vibrator element to enhance the processing under at least one processing step.

A method in accordance with the present invention may comprise a plurality of wash steps wherein at least one of the wash steps is combined with application of vibrations from a vibrator element in order to enhance the washing effect. Alternatively or additionally, a method in accordance with the present invention may comprise a de-paraffϊnization step, wherein the de-paraffϊnization step is combined with application of vibrations from a vibrator element in order to enhance the de-paraffϊnization. Alternatively or additionally, a method in accordance with the present invention may comprise a mixing step, wherein the mixing step is combined with application of vibrations from a vibrator element in order to enhance the mixing. Alternatively or additionally, a method in accordance with the present invention may comprise a mounting step, wherein the mounting step includes an application of a high viscous polymer characterized by application of vibrations from a vibrator element in order to influence the viscosity of the polymer so that the polymer easily may flow and spread over the desired area to enhance the mounting. Alternatively or additionally, a method in accordance with the present invention may comprise a heating step, wherein the heating step is combined with application of vibrations from a vibrator element in order to assist in providing a homogeneous temperature in the sample on the slide. Alternatively or additionally, a method in accordance with the present invention may comprise a Re- Hydration step, wherein the Re-Hydration step is combined with application of vibrations from a vibrator element in order to enhance the R e-Hydration. Alternatively or additionally, a method in accordance with the present invention may comprise a Target Retrieval step, wherein the Target Retrieval step is combined with application of vibrations from a vibrator element in order to enhance the Target Retrieval. Alternatively or additionally, a method in accordance with the present invention may comprise an enzyme application or antibody application step, wherein the enzyme or antibody application step is combined with application of vibrations from a vibrator element in order to enhance the application of the enzyme or antibody.

Within methods in accordance with the present invention, the vibrator is, preferably, arranged to emit acoustic waves of a wavelength adapted to the at least one process that is assisted by the waves. Preferably, the vibrator is arranged to emit acoustic waves of a wavelength that is smaller than or of approximately the same size as the sample carrier and, more preferably, smaller than or of approximately the same size as the sample to be processed. Preferably, a method according to the invention is applied for IHC, SS (special stain), ISH and cytology.

A method of automated sample processing in accordance with another preferred embodiment of the invention comprises the steps of: establishing an automated sample processing system having an automated process operation capability that causes automated process operation events through robotic sample process functions; loading a plurality carriers with biologic samples in the automated sample processing system; loading or accessing data enabling the sample processing system to define at least one protocol for the control of the sample processing of each of the loaded samples; and performing the sample processing using at least one "tapping or knocking" element to enhance the processing under at least one processing step. Preferably, an automated sample processing system in accordance with the present invention may act to accept changes to the system while operating and may automatically adapt to a change in the aggregated events originally scheduled. It also may provide a ^"better approach to adjust how such scheduling may occur as well as providing user opportunities to undo a change such as if its effect would be undesirable. The system may also provide for automatic suggestions to permit an operator to more optimally enhance the schedules on which events occur. Such a system is disclosed in the aforementioned international patent application publication document WO 2004/058404 A2.

As described, sample processing can be accomplished as disclosed herein. In providing this disclosure, it should be understood that the various examples and designs disclosed, for sample processing and other disclosed techniques, are not meant to limit the present invention to any particular embodiment, whether apparatus, method, or otherwise. These descriptions are provided rather to describe various sample processing techniques in a manner in which the present invention can be understood. The descriptions incorporated by reference and the various examples should not be construed to limit the present invention to only such techniques. This disclosure, however, may be understood to incorporate the various techniques in the context of the various embodiments of the present invention.

The techniques and systems of sample processing are addressed in a fashion that may provide the processing of one or more samples or of a plurality of groups of one or more samples in sequential or non-sequential fashion. Processing of samples may be determined by the protocol to be followed for each sample or a protocol for multiple samples. Aspects of the present invention may be especially applicable to sample processing having one or a plurality of processing steps to be performed on one, a portion, or an entirety of samples, such protocols identified in some instances by individual carriers presenting the samples or by the individual samples themselves. As mentioned, the present invention may be especially applicable to immunohistochemistry (IHC) techniques, as well as in-situ hybridization (ISH) and fluorescent in-situ hybridization (FISH), special staining of samples, and micro-arrays; especially techniques incorporating target retrieval or the staining of samples. Furthermore, embodiments may be directed to processing sequences addressing issues of processing control.

Embodiments of the invention may further relate to automated control systems for sample processing and may also be directed to data acquisition, input, maintenance, and retrieval for sample processing, as well as information sharing of processing protocol and processing information, and real-time or adaptive capabilities for processing.

In a preferred embodiment, the invention may comprise an automated sample processing system comprising a plurality of drawers, a plurality of sample carrier elements that may even be each removably configured with one of the drawers, and an adaptive or other sample processing control system. The drawers and sample carriers may be both movable and removable. The sample processing control system may automate the sample processing system such that one or more samples may be processed according to one or more protocols, potentially indicated by information on slides or otherwise input to the system. This sample processing may comprise one or more sampling protocols and steps, such as de- paraffinization, target retrieval, and staining.

A sensor may be provided, in some embodiments, that may automatically identify information from one or more samples, sample carriers, or slides. In embodiments, protocol information may be provided or made available by the sample processing control system. The sample processing system may then process one or more samples or perhaps slides, or one or more batches of slides, concurrently, sequentially, or in any other temporal fashion, potentially in accordance with protocol information previously provided for a sample by a user or other decision maker. This information can then be made available for use by the sample processing control system. Sample batches or individual slides may even be inserted or removed during processing protocol steps by the control and monitoring accomplished by the adaptive sample processing control system.

A preferred method of automated sample processing, in accordance with the present invention, comprises the steps of: accessing at least one of a plurality of samples or sample drawers, providing at least one sample carrier or perhaps a sample carrier retaining assembly configured with at least one sample, configuring at least one of the drawers with the at least one sample carrier, and adaptively processing the sample. The step of processing or perhaps even adaptive processing may be applied to automate the processing of samples and may allow for either or both continuous or batch processing of samples or slides. It may also afford multiple independent sample or slide processing and in some embodiments slide processing to process each slide independently.

Another preferred method of automated sample processing, in accordance with the present invention, comprises the steps of: acquiring or accepting or accessing information such as protocol or reagent information, transmitting such information to at least one sample processing system or even a stand alone processing system, and processing samples. Furthermore, embodiments may provide: for handling, maintaining, sharing, and using the sample processing information. These and other aspects may be provided for individual samples or multiple batch processing, and in a real-time manner. It may also be accomplished in an adaptive manner, perhaps for multiple batch processing or the like.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying figures are incorporated in and form a part of the description, illustrate some of the preferred embodiments of the present invention. Together with the written description and disclosures of the specification, they serve to explain principles of the invention.

FIG. 1 shows a drop of reagent on a sample on a carrier on a support. FIG. 2 shows the drop of reagent after a period of time such that the drop covers all of the sample.

FIG. 3 shows the same a little later, when the reagent has mixed with the sample or is absorbed by the sample.

FIG. 4 shows the implementation of a vibrator within a sample carrier. FIG. 5 shows the implementation of a vibrator within the support. FIG. 6 shows three vibrators within a support.

FIGS. 7, 8 and 9 show alternative positions for a vibrator.

FIGS. 10 and 11 schematically show a carousel instrument.

FIGS. 12 and 13 schematically show an embodiment of an automated processing instrument.

FIG. 14 shows a vibrator embedded in a cover.

FIG. 15 shows a an automated processing instrument in accordance with a preferred embodiment of the invention.

FIG. 16 shows a similar instrument without frame and cover. FIG. 17 is a depiction of an embodiment of a portion of a sample carrier assembly of one embodiment of the invention, i.e. a slide rack in a single drawer.

FIG. 18 is a depiction of an embodiment of a robot arm in the automated processing instrument in FIG. 15.

FIG. 19 shows a flow diagram of IHC processing. FIG. 20 shows a listing of IHC processing.

FIG. 21 is a depiction of a networked embodiment connecting one stainer with one manager and one label printer;

FIG. 22 is a block diagram showing some of the internal software features;

FIG. 23 is a depiction of an embodiment connecting multiple stainers with multiple managers and multiple label printers; and

FIG. 24 is a depiction of an embodiment connecting a system to a lab network and lab information system.

FIG. 25 shows a preferred embodiment of a SAW-rack installation in an Autostainer. FIG. 26 shows schematically the principles of SAW mixing on a microscope slide.

FIG. 27 shows a light micrograph of cytokeratin (brown) staining of a mamma carcinoma without SAW mixing.

FIG. 28 shows a light micrograph of cytokeratin (brown) staining of a mamma carcinoma after SAW mixing.

DETAILED DESCRIPTION The following descriptions are provided to describe various embodiments of the present invention in a manner to facilitate a more detailed understanding some of the inventive features. The variously described examples and preferred embodiments should not be construed to limit the present invention to only the explicitly described systems, techniques, and applications. This description may further be understood to incorporate the various systems, techniques, and applications, both singularly and in various combinations consistent with the various inventive features and embodiments of the present invention. Accordingly, the following is a detailed description of a number of specific embodiments of the invention. To more particularly appreciate the features of the present invention, the reader is directed to FIGS. 1-28, to be examined with reference to the following discussion.

Each of FIGS. 1-9 shows a microscope slide with a biological sample to be processed and one of a number of various positions for a vibrator element generating a vibrating movement. FIG. 1 schematically shows one embodiment of an arrangement of a carrier 10, such as a microscope slide and a sample 12 on the carrier 10 in close contact with a support 15. A droplet 14 of a reagent is applied to the sample. In FIG. 2, the reagent 14 has spread over most of the sample, and in FIG. 3 most of the reagent has mixed with or is absorbed by the sample. The process indicated in the FIGS. 1-3 may be accomplished in many ways. According to the present invention, the sample is vibrated in a short period such as a few seconds or minutes, depending on the properties of the sample and the reagent or the probe that has to be added to the sample. More specifically, the viscosity of the reagent has a great influence on the time needed to obtain the desired reactions. By vibrating the reagent or any other substance, which has to be added to the sample, the assay time may be considerably reduced. Further experience has proven that several other benefits may be obtained, such as higher sensitivity and better reproducibility. These advantages are possibly due to a more homogeneous mixture of sample and reagent that is accomplished through the vibration of the sample and reagent.

The vibrations may be generated in several ways. Two such ways of generating vibrations are: (1) by use of a robotic arm, tapping (knocking lightly) on the top of a slide, as indicated by the solid arrow A and (2) by the use of a robotic arm, tapping (knocking lightly) on the edge of a slide, as indicated by the dashed arrow B. The vibrations may be produced by at least one embedded vibrator element 16, such as a piezo crystal in carrier 10 as shown in FIG. 4, or in the support 15, as shown in FIGS. 5-9, or in a cover above the sample, as shown in FIG. 14. It may be advantageous to incorporate two or three such vibrator elements 16 in each support 15 as shown in FIG. 6, in order to ensure a homogeneous agitation of all of the sample and reagent on a slide. Preferably the vibrator elements 16 are SAW-chips, generating surface acoustic waves.

A similar set-up is illustrated in Fig. 26. showing schematically the principles of SAW mixing on a microscope slide. SAW chips below the slide generate ultrasound microwaves that propagate through the medium generating circular waves when they reach the surface of the water droplet on the slide. A coupling liquid between the chip base and the glass slide is necessary in order to allow transduction of the mixing waves. The coupling liquid maybe any liquid applied on the slide (buffer, water etc.) and is automatically drawn into the narrow space under the slide by capillary forces.

The vibrator elements 16 illustrated in FIGS. 4-9 and 26 may be connected through conducting wires (not shown) to a power supply such that the power supply is controlled from a controller system providing control of all processing in the automated sample processing system. More specifically, the vibrations applied to any sample during a processing are, preferably, controlled according to the processing protocol for that specific sample. Thereby, it is ensured that each sample is processed specifically as requested.

One aspect of the invention focuses on the use of vibrator elements to improve the performance of an automated staining apparatus and a method of automated treating of samples. As to this aspect, the present invention relates to an automated staining apparatus for treating samples arranged on carrier elements or means, such as but not limited to microscope slides, located at defined positions close to or in the apparatus by removing a portion of selected reagent from a station containing a plurality of reagents and thereafter applying the reagent to a sample, e.g. a tissue, organic cells, bacteria etc., arranged on the carrier means. This aspect of the invention facilitates that two or more reagents are mixed and the mixture applied to a sample. It also relates to a method of automated treating of samples by mixing reagents and applying the mixture to the samples.

Staining apparatuses for staining and treating samples by means of a probe normally comprises a first station for containing one or more reagent vials; a second station for mounting slides, a probe arranged for removing a portion of reagent from a selected reagent vial and applying the reagent to a slide on which the sample is arranged and a drive means for moving the probe between the various stations.

An object of this aspect of the present invention is to improve the known apparatuses for staining samples as well as the method for automatic staining of samples by facilitating a wider range of available processes or procedures used to implement treatment, so as to ease the implementation of different staining and/or treatment processes that may be performed automatically, alternatively or additionally to provide an increased quality of some specific staining processes.

The term staining is used for the end product of the process, by which certain parts of the sample may be stained, i.e. has obtained a different colour, either in the optic range or in another electromagnetic range, such as ultra violet, or the staining may be an detectable, preferably automatically detectable, change in properties, such as fluorescent properties, magnetic properties, electrical properties or radioactive properties. To obtain the staining, the sample normally has to undergo a series of treatment steps, such as washing, binding of reagents to the specific parts of the sample, activation of the reagents, etc. and each treatment step may include a plurality of individual treatments.

In some staining processes, it may be required for one or more treatments to use a mixture of reagents prepared from two or more separate reagents which may be somewhat incompatible e.g. unmixable, such as a water based and an oil based reagent, or insoluble, and therefore requires that the two or more reagents are manually prepared and introduced into a reagent vial shortly before starting the staining process in order to obtain the best possible staining result for the selected examination purposes. For other processes, different staining process steps require a mixture of the same two reagents but in different dissolved ratios. Some process steps require mixtures of two or more reagents that, when mixed, have a limited time window of usability because internal chemical processes deteriorate the mixture. By providing a staining apparatus having an automated mixer integrated therein, these types of staining processes can be performed automatically instead of requiring human interaction or manual performance of some process steps in a much more automated process, and the quality of the staining process may be improved as a desired degree of mixing of reagents may be provided or an optimal application time window for a deteriorating mixture may be reached.

The term target retrieval as used in this description may include antigen retrieval, epitope retrieval, Protelytic enzyme target retrieval, Heat induced antigen retrieval (HIAR), demasking etc. To facilitate the specific recognition in fixed tissue, it is often necessary to retrieve or unmask the targets, i.e., the biological markers of interest, through pre-treatment of the specimens to increase reactivity of the targets. This procedure is referred to as "antigen retrieval", "target retrieval" or "epitope retrieval", "target unmasking" or "antigen unmasking." An extensive review of antigen retrieval (antigen unmasking) may be found in Shi et al. 1997, J Histochem Cytochem, 45(3):327. Antigen retrieval includes a variety of methods by which the availability of the target for interaction with a specific detection reagent is maximized. The most common techniques are enzymatic digestion with proteolytic enzymes such as e.g. proteinase, pronase, pepsin, papain, trypsin or neuraminidase in an appropriate buffer. As an alternative or in combination heat induced epitope retrieval (HIER) may be applied using sources of heat such as e.g. microwave irradiation, heating in a water bath, a steamer, a regular oven, an autoclave or a pressure cooker in an appropriately pH stabilized buffer, usually containing EDTA, EGTA, Tris-HCl, citrate, urea, glycin-HCl or boric acid. Detergents may be added to the HEER buffer to increase the epitope retrieval or added to the dilution media and/or rinsing buffers to lower non-specific binding. The antigen retrieval buffer is most often aqueous, but may also contain other solvents, including solvents with a boiling point above that of water such as e.g glycerol. This allows for treatment of the tissue at more than 100 oC at normal pressure. Additionally, the signal-to-noise ratio may be increased by different physical methods, including application of vacuum and ultrasound, or freezing and thawing of the sections before or during incubation of the reagents.

Endogenous biotin binding sites or endogenous enzyme activity (for example phosphatase, catalase or peroxidase) may be removed as a step in the staining procedure, e.g., endogenous biotin and peroxidase activity may be removed by treatment with peroxides.

Endogenous phosphatase activity may be removed by treatment with levamisole.

Endogenous phosphatase and esterase activity may be destroyed by heating.

Blocking of unspecific binding sites with inert proteins like, HSA, BSA, ovalbumin, fetal calf serum or other sera, or detergents like Tween20, Triton X-100, Saponin, Brij or Pluronics may be used. Blocking of unspecific binding sites in the tissue or cells with unlabeled and target unspecific versions of the specific reagents may also be used.

The carrier elements or perhaps means are preferably arranged in groups or series on trays or the like, so that a plurality of carrier means may be removed from or situated in the apparatus simultaneously, and the apparatus preferably also comprises means for performing the intermediate storage of the carrier means with samples thereon and the removal of the carrier means from the apparatus automatically.

The operation of the staining apparatus may generally be controlled by means of a control element or perhaps a control means, typically a computer having a central processing unit and one or more memory units associated therewith, an control element or perhaps a means for controlling the various operations of the apparatus by controlling stepper motors, solenoids, valves and/or other drive or control parts of the apparatus. The control means may have one or more data communication ports for enabling data communication with external computers by wire or wireless. The control means does not have to be physically arranged within the apparatus itself but may be a computer external to the staining apparatus and connected to the apparatus via a data transmission port thereof.

The operation of the staining apparatus will generally be controlled by means of control means, typically a computer having a central processing unit and one or more memory unit associated therewith, means for controlling the various operations of the apparatus by controlling stepper motors, solenoids, valves and/or other drive or control parts of the apparatus. The control means may have one or more data communication ports for enabling data communication with external computers by wire or wireless elements. The control element or perhaps means does not have to be physically arranged within the apparatus itself but may be a computer external to the staining apparatus and connected to the apparatus via a data transmission port thereof.

According to the present invention, the vibrator element(s) 16 (FIGS. 4-9, FIG. 14) are incorporated into an automated sample processing system comprising a plurality of positions for sample carriers 10. Such systems may have the sample carriers arranged on a carousel as indicated in FIGS. 10 and 11. Other, alternative systems may comprise a plurality of separate positions arranged in a compact rotary symmetric unit comprising several levels (floors) 9 of positions enclosing or adjacent to a neighboring transport means 7 that is able to elevate and insert a sample carrier to a specified position on a specified level by a translational movement. A schematic sketch of such configuration is illustrated in FIGS. 12-13. In a further embodiment a sample processing system according to the invention may comprise a plurality of drawers 110, FIG. 15, used for the handling and processing of samples and sample carriers such as slides potentially microscope slides. Other sample carriers may be accommodated consistent with the present invention. Each drawer may be configured to accommodate carrier-retaining assemblies that hold one or, most likely, a number of the particular carriers, slides, or samples involved. In holding slides, the carrier retaining assembly serves as a slide retaining assembly 106. There may also be carrier racks, modules, or magazines encompassed within each of the two broad terms. As one embodiment of a sample carrier retaining assembly, a slide retaining assembly 106 is shown in FIG. 17. The slide retaining assembly, and indeed the generic carrier retaining assembly may comprise a slide rack, module, or a number of magazines.

FIG. 25 shows as an example an experimental set-up of a SAW-rack installation in an Autostainer from Lab Vision Corporation. Three SAW chips are imbedded in each of twelve aluminium blocks, each designed to support a microscope slide. A microscope slide has been placed in position four and a spirit level is placed in position five to ensure proper levelling. To prevent the fluids from being "pushed off the slide it is important that the slides are accurately levelled when SAW mixing is turned on. Examples:

Anumber of tests were carried out using the set-up shown in FIG. 25. The method was tested with four different SAW chips, with different mixing properties installed on a modified Autostainer (AS) rack.

The SAW chips were delivered by Advalytix, (Brunnthal, Munich, Germany). The SAW chip setup included the following four different chips-designs for two different frequencies and 4 different power values:

Frequency: 125MHz and power: I) 125mW and II) 400mW and

Frequency: 80MHz and power III) 250mW and IV) 500mW The SAW chips may be used with continuous operation or pulsed with various duty cycles. The frequency may be sweeped around the center frequency of 80 MHz and 125 MHz respectively. Sweeping the frequency may provide a more homogenious agitation all over the slide and samples.

The 80MHz - 500 mW SAW chip appeared to perform optimal in all tests. The main conclusions of this study are: A reduction in the primary antibody incubation time by at least 50% with

SAW mixing is broadly applicable irrespective of current incubation times.

Dilution of primary antibodies by a factor 2-4 can be compensated by SAW mixing.

A reduction of primary antibody incubation time by 50% is achievable with active SAW mixing only 13% of the incubation time, e.g. applying pulsed operation of the SAW chip with a duty cycle of about 13 %.

When applied during visualization steps (both HRP and AP) SAW increases staining intensities. However, the effect on visualization is not as pronounced as when applied during primary antibody incubation. As an example of the results obtained FIG. 27 and 28 show two light micrograph of cytokeratin (brown) staining of two neighboring slices of a mamma carcinoma. The two Figures represent sequential sections from a microtome preparation of a single tissue sample of the mamma carcinoma. In Fig 27 the sample section was subjected to the standard procedure including 30 min. incubation with primary antibody and no SAW mixing. In Fig 28 the sample was subjected to a new procedure according to the present invention including only 15 min. incubation with primary antibody and continuous SAW mixing (80MHz / 500 mW). Please note that there is no nuclear counter-stain with haematein.

Reduction of the primary antibody incubation time with 50% Based on the preliminary results obtained by manual tests on a SlideBooster from

Advalytix the basic test object was to verify that SAW mixing applied in an automated instrument could compensate for a reduction of 50% in primary antibody incubation time.

Using a cytokeratin antibody and a testing protocol that are particularly sensitive to changes we reduced the incubation time from 30 to 15 min. and compared the slides. IHC scores were consistently found to be higher for slides that were incubated 15 min. with continuous SAW compared to the 30 min. control (see Figure 3).

To substantiate these findings a number of antibody protocols have been tested: CD3: Continuous mixing, incubation reduced from 20 to 10 min. IHC scores higher than reference was observed with 80MHz/ 500mW.

MIB-I: Continuous mixing, incubation reduced from 30 to 15 min. IHC scores higher than reference was observed with 80MHz/ 500mW.

Her-2 (HercepTest): Continuous mixing, incubation of primary antibody reduced from 30 to 10 min. To push the system even further all slides were only incubated in visualization reagent for 10 minutes (normally 30 min.) as well. Surprisingly, no difference was observed between tissues or control cells incubated for 30 or 10 min (primary antibody). SAW treated slides were not scored higher than controls.

The following tests were only done using the optimal chip setting (80MHz / 500mW). ER (PharmDX kit): Continuous mixing, incubation reduced from 30 to 15 min. SAW treated slides matched reference.

CD45: mixing on for 12.5% of the time, incubation reduced from 10 to 5 min. SAW treated slides matched reference. Vimentin: mixing on for 16.6% of the time, incubation reduced from 10 to 5 min.

SAW treated slides matched reference.

CD34: mixing on for 16.6% of the time, incubation reduced from 15 to 7 min. Since reference incubated with no SAW for 7 min. was equally intense as tissue incubated for 15 min. we were not able to register any effect of SAW. Later studies will determine if incubation can be reduced to 5 min.

In conclusion, SAW mixing is able to reduce the time of primary antibody incubation with 50% or more for a broad range of antibodies and it seems likely that incubation time can be shortened even further for some of them. Results obtained with the application of SAW mixing for a fraction of the total incubation time should be regarded as an attempt to find a realistic SAW setting in order to know what the limitations of the system are.

Dilution of the primary antibody

Another approach to test the effect of SAW mixing on IHC performance is to see whether a dilution of the primary antibody can be compensated by SAW mixing.

Two N-series antibodies CD3 and MIB-I were diluted 1:2; 1:4 and 1:8 (no change in protocol incubation times) with and without SAW. SAW was able to compensate for a dilution of 1:2 of the CD3 antibody and 1:4 of the MIB-I antibody. At these dilutions the antibodies were assigned a score scored equal to, or higher than, references incubated with the normally diluted antibody.

In conclusion, SAW mixing can compensate for dilution of the primary antibody in the 1:2 — 1:4 range with individual dilution limits for each antibody. In conclusion, we find that it is possible to reduce the effective time with SAW mixing to around 13% and reduce the primary incubation time by 50% for a number of antibodies, but this effect can only be achieved with the 80MHz 500mW chip.

The present invention is specifically advantageous when combining the assay-time- reducing vibrator element and the scheduling feature known from the aforementioned international patent application publication WO 2004/058404 A2. The event scheduler as disclosed in the publication WO 2004/058404 A2 is able to provide a scheduling of the many events that has to be accomplished for the plurality of slides, e.g. 64 slides where each slide may be scheduled for about 30 steps as described in FIGS. 19 and 20. In order to benefit from the reduced assay times, it is essential to apply a scheduler that is able to schedule the various steps in the most time efficient manner, and still maintain the specifications for the processing as stated in the many protocols, one for each of the many slides.

One object of the present invention is to provide a staining apparatus and a method for automatic staining of samples, in which the total process time for completing or even entering the staining protocol may be reduced. In particular, it is an object of this aspect of the invention to reduce the amount of time needed in general. To this end at least one vibrating element is arranged for transferring vibrations to the sample and any reagent in touch with the sample. The vibrating element may be a piezo electric element, but several other vibrators may be applied. The vibrator may be arranged in the sample carrier - such as in a microscope slide, in a support for the sample carrier or in a neighbouring element able to touch the sample carrier. Alternatively the vibrating element may be located in a specific position whereto the slide to be vibrated is moved.

In a preferred, embodiment, a piezo electric element 16 (FIGS. 4-9) is arranged in a supporting member 15 able to support the sample carrier10. The piezo electric element 16 is connected to a voltage supply (not shown) controlled by the protocol for sample processing of the specific sample on the sample carrier. Preferably at least one piezo electric element is arranged in a support means for each slide in a slide retaining assembly 106 (FIG. 17). In the preferred embodiment the piezo electric element is arranged in the same supporting member as heater means arranged to control the temperature of the slide.

Preferably, the vibrator elements are incorporated in all positions for sample carriers, and able to be actuated for a period during all processing steps. However, it is preferred that the vibrations shall only be on for short periods. FIG. 20 shows a long series of various steps typical for a staining process. A short vibration of a few seconds may be advantageous to almost all of the steps. Only in the staining step — specifically during the DAB step in a "Chromogen/Counter stain treatment" — vibrations might deteriorate the staining, or at least influence the staining in such manner that the result might differ from what a trained pathologist can recognize. Even in some steps, such as Eosin & hematoxylin, during the staining vibrations might improve the staining result.

During target retrieval a brief vibration in the beginning of the step and possibly repeated in brief periods might assist in providing a homogeneous temperature of the sample. It is not recommended to vibrate all the time during the full step. Generally it is recommended to vibrate during 3-5 seconds of a step and turn the vibrator off for about 60 seconds. And then repeat this sequence a few times.

In steps comprising change of buffer, such as the de-paraffinization and re-hydration steps, the original times of about 5 minutes shown in FIG. 20 may be reduced to about 1 minute or even less. Also enzyme blocking and enzyme treatment (proteolyse) steps may improve when using vibrations. Antibody and visualization steps will also benefit from vibrations by providing better signals.

Generally the number of wash steps might be decreased by the use of vibrations. This is due to the fact that a "carry over" from one step to another may be less if the mixing is perfect on the slide for every step. The wash will become more efficient. The improvement may be considerably when using polymers that diffuse slowly. Hybridization efficiency is very dependent on probe concentration; accordingly vibration may highly also improve hybridization by improving the mixing of probe and sample. As it appears from the foregoing a lot of time may be saved in each of the processing steps. However to really benefit from this in an automated processing instrument processing e.g. 64 slides, each due for about 30 different steps, wherein some of the steps require a robotic arm moving to pick up few micro-liters of a specified reagent and then moving to the position of the slide having a sample to be treated with that reagent. Such movement of a robotic arm takes time and all of the slides shall be treated with a reagent, and preferably to a specified time after a previous step. The scheduling is a highly complicated task when about 30 different steps have to be scheduled for 64 slides with different protocols in the most optimal manner, i.e. in such manner that all slides are processed as fast as possible.

A presently preferred embodiment of automated sample processing system in which a vibrator element, as described above, may be advantageously employed is illustrated in FIGS. 15-18 and is described below in details. Further aspects and details of this preferred automated sample processing system are provided in the following international patent application publications, each of which is incorporated by reference herein in its respective entirety: international patent application publication WO 2004/057307 Al, international patent application publication WO 2004/057308 Al, international patent application publication WO 2004/058950 Al, international patent application publication WO 2004/059287 A2, international patent application publication WO 2004/058404 A2, international patent application publication WO 2004/059284 A2, international patent application publication WO 2004/059288 A2, international patent application publication WO 2004/059441 A2, and international patent application publication WO 2004/059297 Al.

FIG. 15 shows a top view of a sample processing system 101 in accordance with the present invention. The sample processing system 101 is configured, to achieve an appropriate sequence of events that achieves a desired result to some degree. In achieving this sequence in an automated fashion to some degree, the sample processing system is deemed an automated sample processing system and achieves automatic processing of at least one sample. This automated sequence as well as other aspects of the invention may be controlled by hardware, software, or some combination of them to accomplish a desired sequence with limited human intervention. Regardless of how achieved, the automated control may be provided by a process operation control system to direct the various activities. As shown in FIG. 22, this (as well as other functionalities discussed) may be software programming or subroutines; again, it may also include hardware or the like. The sample 198 (FIG. 17) processed may be any material, but is most likely a biologic material such as a biologic al sample or a biological specimen, perhaps such as a histological sample, e.g. tissue and cell specimens, cells, collections of cells, or tissue samples, the definition to include cell lines, proteins and synthetic peptides, tissues, cell preps, cell preparations, blood, bodily fluids, bone marrow, cytology specimens, blood smears, thin-layer preparations, and micro-arrays. It should also be understood to include slide-based biological samples. As used, a sample may be arranged on a carrier element 197 (FIG. 17) such as a slide, or microscope slide, or the like that may maintain the sample's position or integrity. The carrier element 197 may be configured to move and thus reposition the sample 198. As such, it may be considered a movable carrier element. In processing a slide, the automated sample processing system may serve as an automated slide processing system.

The automated sequence may involve a significant number of steps. In fact, each process can itself require many automated movements to achieve its goal. Each of these types of operations or actions may be relevant to understanding an instrument's operation. Further, each of these types of operations or even a lesser set of significant events may be considered important details of the sample process operation. As explained later, it may be valuable to capture information relative to a significant number of these actions such as all of these operations, some subset of these operations, one-half of these operations, one-third of these operations, or the like. Further, even the nature or type of the events that may be of interest may be varied. In general, any event that may indicate the propriety of operation or processing may be a subject. Naturally in order to achieve automated processing it will be necessary to schedule the various sample processes or process operations desired. This can be achieved by an item of software or the like that acts as a multiple event scheduler 401 (FIG. 22). A particular design of a system may include cabinet sections 102 (FIG. 15) that may form outer portions of the system and serve to address general structural considerations of the system (note that a top cabinet section is not shown in FIG. 15).

The slide retaining assembly 106 in FIG. 17 may be configured to accommodate a plurality of slides in at least one configuration in corresponding sample carrier retention devices 108. The sample carrier retaining assemblies are utilized in the processing of samples as further described below. It should be further noted that the sample carrier retaining assembly can be removably configured with the drawers 110, and may be stackable or nested within other retaining assemblies.

The general sample processing system 101, and even one or more drawers 110 in the sample processing system 101 may accommodate processing materials such as reagent containers 199 for sample processing, also further described below. A processing material retaining assembly, such as a container rack 111, shown in FIG. 15, may be utilized to accommodate reagent containers 199 (FIG. 16) or other processing materials within each of drawers 110. These drawers may be lockable through programming to prevent access during certain stages of automatic operation. Bottle inserts may be preferably configured with the retaining assembly to ensure proper processing material positioning within the processing material retaining assembly and the drawer.

Multiple drawers 110 may be included to allow for one or a plurality of sample processing protocols to be performed by the system 101. Past efforts at sample processing, as previously described, may have been limited to processing sequences for an entire batch of carriers within the system. The present invention, however, in part by providing a plurality of drawers and carrier retaining assemblies, may allow for individual, batch, or multiple batch processing, including real-time or adaptive capabilities, as further described below. The vibrator elements may be incorporated in positions in a single drawer specifically arranged for samples to be processed as fast as possible, or in any number of drawers, and preferably in all drawers. Indicator elements 112 (FIG. 15) may be provided to indicate a status and accessibility of the drawers and the carriers or materials within each drawer 110 for an operator of the system. In one embodiment, visual indicators, such as light emitting diodes in preferred embodiments, may be used to indicate if a drawer is available, and perhaps unlocked, during operation of the sample processing system, and may indicate conditions such as a locked or open condition of a corresponding drawer, carrier capacity status of the drawer or of a carrier retaining assembly within the drawer, and chemical inventory status of the sample processing system, such as reagent loading status or capacity. A warning indication may be given by these or other indicator elements, as well as other indicative signals. One or a plurality of sensors may be utilized to determine the status of the drawer as indicated by the indicator elements 112 and to further provide processing status as further described below. Thus the system may provide at least one substance in a lockable reagent retaining assembly. Interestingly, the lockable reagent retaining assembly may be established as being generally in an unlocked state, perhaps the majority of the time the system is operating, the lockable reagent retaining assembly may be unlocked such that an operator may access that drawer. This may also exist during a significant portion of the time the system is operating, perhaps even such as for 75% of such time. In this manner the system may be considered as providing a generally unlocked reagent retaining assembly.

A processing material unit may be utilized to provide various processing material to the sample processing system 101 and to afford the hazardous and non-hazardous segregation of waste produced during sample processing and the avoidance of cross-contamination. In one embodiment of the present invention, the processing material unit may be configured to accommodate one or a plurality of containers such as de-paraffinization solution or other material utilized in sample processing. In some embodiments, the unit may also accommodate waste containers to provide for the collection of waste material from the sample processing. Tubing or other fluid transmission elements may be connected with the containers and the sample processing system 101. Tubing or other fluid transmission elements may also be connected with the waste containers and the system 101. The present invention further relates to the use of an apparatus of the present invention as described above for exercising the method of the present invention. The embodiment shown in the figures and described in details below is only an example of an apparatus in accordance with the present invention and is not limiting the wider scope of the invention as described in the enclosed claims.

As shown in FIG. 16, a detailed description of one embodiment of this aspect of the invention involves staining apparatus 201. The staining apparatus 201 may comprise a rectangular frame 204 surrounding a first station 202 comprising an array of compartments wherein each compartment a reagent vial 203 is placed, and a second station 205 wherein a number of separate racks 206 is placed, and where each rack may comprise a number of slides 207 mounted side by side in the rack 206. In the embodiment shown, each rack may hold up to 8 slides, but the rack may be designed to hold any suitable number of slides. With eight racks arranged side by side, the shown embodiments may hold up to 64 slides 207 each having a sample, e.g. a tissue mounted on the upper side of the slide, so that reagent may be applied from above to the sample on each slide.

A robot arm to move a probe 210 in X and Y direction as indicated by the arrows X and Y may be arranged above the frame 204 of the staining apparatus. The robot arm may therefore position the probe 210 above all reagent containers 203 as well as above all the slides 207, and may further operate the probe 210 to remove portions of a reagent contained in any of the containers 203, to transfer the portion of reagent and apply it to any of the slides 207 in order to provide a selected staining or treatment of the sample on each slide 207. By use of a suitable control element, e.g. a computer having the appropriate software, subroutines, or input data for the purpose, this staining apparatus 201 may be able to automatically stain or treat samples requiring different staining or treatment reagents and processes.

Having the appropriate input data, the control element or perhaps means of the apparatus may operate the robot arm to commence a staining or treatment run by moving the probe to a first reagent container 203, into which the probe tip is inserted and liquid is aspirated up into the probe 210 in an amount corresponding to the number of samples to be stained or treated, in accordance with the input data provided to the control element. Additionally, under certain conditions, the instrument may be required to perform a reagent inventory before a staining or treatment run can commence. This inventory may be accomplished by use of the probe tip to actually touch the liquid surface in each reagent vial 203. To prevent cross-contamination between the reagents in the various containers 203, a cleaning of the probe 210 or at least the probe tip may be required after each measurement of a reagent level.

The probe 210 may be moved by the robot arm towards the slide retaining assembly

205 in which the slides 207 are mounted. The slides 207 may be situated with the surface horizontally oriented and the probe 124 may dispense the required amount of reagent on the appropriate slides in accordance with the input data. Alternatively, the probe 124 may be moved by the robot arm towards the reagent mixer 209 where it may release reagent into the cup of the reagent mixer 209, and may be subsequently moved to the probe washing station 208. The robot arm may move the new clean probe to a second selected reagent vial 203 for collecting a selected amount of reagent from the second vial 203, and the probe may thereafter by means of the robot arm be moved to the reagent mixer 209, where the reagent in the probe 210 may be released into the cup of the mixer containing the first selected reagent. This may be commenced several times if more than two reagents are to be mixed for a specific staining or treatment process.

In accordance with the desire for an automated processing system, embodiments of the present invention may include robotic sample process functions or a robotic motion system 172 (FIGS. 16, 22) responsive to the process operation control system 171 (FIG. 22) to achieve the desired operation steps. This may further comprise an arm 120 (FIG. 15) utilized in sample processing, potentially having robotic movement, and in some embodiments, Cartesian movement. In this manner, the system may provide an automated process operation capability that causes automated process operation events through robotic sample process functions. These may be responsive (a term intended to encompass any interaction, whether connected or not and whether directly or indirectly having an effect) to a number of perhaps stand-alone devices such as stand-alone stainers. The arm 120 may comprise (FIG. 18), in some preferred embodiments, one or more elements, such as an actuator probe 122, a syringe or probe 124, a sensor element 25 and a non-discrete or other volume fluid and/or air applicator. The actuator probe may be utilized in the configuration and. manipulation of the carriers in sample processing, further described below. In some preferred embodiments, the actuator probe 122 configures and manipulates the configuration of slides in the sample carrier retention devices 108 by actuation of carrier adjustment element 130 (see for example FIG. 17), and in some embodiments, by contact with the slides. As mentioned, in some embodiments, manipulation or movement of the slides or the samples may be accommodated. This movement may result in a horizontal or vertical configuration of the slides to facilitate sample processing as described below. An actuator probe may be used for tapping on a slide in order to generate vibrations in the sample and reagent. Further a vibrator element may be incorporated in the actuator probe.

As mentioned above, there may be a large number of process steps accomplished. As may also be appreciated from the nature of the processes envisioned, there may be uses of many different substances or the like. Whether involving a substance or merely a physical action, these types of items may be considered as relating to operationally-influential exteriorly-consequential information. The item may be operationally-influential in that it either its operation or failure in operation may directly or indirectly influence some type of conduct. This conduct may be exteriorly-consequential in that it may be a conduct that does not take place within the process system itself but external to it. As such the present invention may provide the capability to monitor that information. This capability may even be considered as an operationally-influential exteriorly-consequential information monitor 402 as shown generally in FIG. 22. Thus, the present invention may include an ability to monitor information of a broad nature.

As but one example, the present invention may involve monitoring exteriorly- consequential information that is actually operationally-altered outside information in that the activity conducted as part of the process system's operation actually causes a change in the information. But one example of this might be using up a particular stain substance. By monitoring this category of information, the present invention may be considered as monitoring operationally-altered outside information. This embodiment may thus be considered as including an operationally-altered outside information monitor. Of course, these events may be influenced at least in part by at least some of the robotic sample process functions.

As previously mentioned, arm 120 may comprise syringe 124. The syringe 124 may be considered a probe in some embodiments, depending upon the requirements of protocols to be performed. Syringe 124 may be fluidically connected with and may apply one or more of the following: rinse agents, such as water; containers, potentially removably fluidically connected for the aspiration of reagents, such as aspiration of reagents from containers and to the samples presented with the carriers; and blow off or other removal agents such as an air source. Syringe 124 may be utilized to pierce processing material containers such as reagent containers. In some embodiments, a reservoir may be provided with the arm 120 to allow for various volumes to be aspirated by syringe 124. The unique configuration of the reservoir allows for efficient cleaning and drying of the internal portions of the syringe while allowing for the accurate pipetting or otherwise aspiration of a wide range of volumes.

Arm 120 may, in some preferred embodiments, comprise a sensor element 25. The sensor element may be used to automatically determine location and other status information of components of the sample processing system, such as reagent containers, or other processing material containers, or sample carriers. This may be used to teach the system proper and/or actual locations, and to calibrate, self-calibrate, or self-align the system, or the like.

In preferred embodiments, the sample processing system 101 may include an automatic slide identification element. This may be controlled to achieve the act of automatically identifying a plurality of slides. This may also be more generic such as there may be some type of sensor element and it may even comprise a reader or scanner, such as a CCD camera, utilized to determine status information of processing materials, such as reagents as well as to identify slides. The sensor element, for example, may read, detect, or otherwise determine information in the sample processing system 101, for example, from processing material containers, such as, for example, reading coded or perhaps encrypted information provided on the container to determine reagent type and reagent location within the system. The sensor element may also determine status information of sample carriers. For example, in some embodiments, slides configured with a slide retaining assembly may be provided with informational indicia, such as a code, that may indicate information about the sample presented on the slide or the processing protocol to be performed. The sensor element may read the code of the slide to determine the protocol to be performed for the particular slide and sample.

A cleaning station 140, shown in FIG. 15, may be included to clean elements of arm 120, and in preferred embodiments, may function to clean or otherwise remove completely the previously deposited reagent from the probe, or remove elements containing the internal and/or external surface of the probe and/or syringe 124. In one embodiment, the cleaning station may be configured to clean elements of arm 120, such as syringe 124, while such elements are configured with arm 120. Trie syringe may be cleaned, for example, with a water rinse through the syringe while the syringe is positioned at the cleaning station. In other embodiments of the present invention, the cleaning station 140 may be configured to allow a drop off and pick up of elements such as syringes for cleaning while allowing the processing throughput of the sample processing system to continue.

In some embodiments, multiple probes or syringes may be used to apply fluids required for the staining of histological tissues samples mounted or otherwise presented on slides. This may encompass automatic staining accomplished through a slide stain element such as the items included on the robotic motion system 172 discussed above. The sample processing system may drop off a "dirty", contaminated, or used probe or syringe and swap it for a "clean", uncontaminated, sterilized or an unused one. One or more probes or syringes may be cleaned while the system continues processing of samples, such as applying reagent or stain with an alternate probe or syringe. In addition, or alternatively, the probe used may be washed while attached to the robotic motion system. The system 101 may access, use and wash multiple probes or syringes for pipetting or otherwise aspirating fluids required for the staining of samples mounted or otherwise presented on slides. To eliminate cross contamination, a system with a single reusable probe may wash the probe between each fluid applied. The task of washing the probe can have a large impact on the throughput of the overall system. The present invention may allow for multiple probes to be available to the system for use. The system may continuously have a clean, uncontaminated, sterilized, or an unused probe available to use and sample processing is not impacted by the required cleaning routine. The cleaning routine may be necessary to eliminate the possible cross contamination of fluids and, in some embodiments, may take up to about 1 minute to accomplish. The cumulative impact of the cleaning routine on a series of processing steps can add time to the throughput capabilities of the system. The addition of multiple probes or syringes may eliminate this impact and significantly decreases the time required to process the samples.

Embodiments of the present invention may comprise a mixing station 150, shown in

FIG. 15. The system may mix component fluids, such as dyes, buffers, or other processing materials, preferably on demand and as the processing steps and protocols dictate. Fluids required during the processing steps may sometimes need to be mixed with other fluids to create a final activated fluid mixture or cocktail. However, the activity levels of these mixtures can be time sensitive and may therefore only be effective for a short period of time. The on-demand, or perhaps just-in-time mixing of fluids is advantageous in that it allows the fluids to be mixed immediately before being used. This may be coordinated with the scheduling functions discussed below to permit the simultaneous goals of on-demand mixing with enhanced scheduling. The syringe or probe 124, in preferred embodiments, will aspirate fluids into and from the mixing station 150 to mix component fluids. A rinse may further be dispensed into the mixing station to sterilize the station.

In preferred embodiments, slides are movable and configurable in both vertical and horizontal positions as required for the pretreatment and staining process. This allows for the automation of the pretreatment and staining of slides in various manners, including pretreatment and staining as accepted in conventional manual laboratory methods. The slides are initially loaded into the carrier retention assemblies, such as slide racks, and drawers in the horizontal position. If pretreatment is required, such as de-paraffinization, the system rotates the slide into the vertical position and lowers these samples into a processing tank, further described below, filled with the required fluids. In some embodiments, the slide rack is lowered to affect lowering of the slides. To perform the staining process on the slides, as described below, the system rotates or moves the slide to the horizontal position and a syringe or probe applies fluid to the sample, providing a horizontal staining of the sample. Each slide can be rotated independently allowing for the independent processing of different samples with different requirements.

The system automates and, in some embodiments, mimics or otherwise corresponds to the procedure and physical attributes of the supplies used manually to perform these same pre-treatment processes. Accordingly, a processing tank may be provided. In some embodiments, components of each processing tank may be configured within a drawer 110. In some preferred embodiments, the fluids volume needed to perform pre-treatment processes are maintained but instead of the slide orientation with each other being face-to- face, as in conventional systems, they are side-to-side, although other slide configurations are not disclaimed. The processing tanks provide even distribution of fluids across the face of the slide.

In some embodiments, the processing tanks have the ability to heat and cool the slides. Heat may also be applied to each individual slide by a thermal device. The precision and physical application of the temperature control can result in standardization and repeatability of process steps. Filling and heating tasks are performed by a computer controlled scheduler, as further described below. Fluid volume may be adjusted to account for the presence or absence of any number of slides.

In some embodiments, the individual fluids used for pretreatment may be contained in the system cabinet. De-paraffinization fluids (except DI water) may be drawn into the processing tanks, then returned to their containers for reuse. On a periodic basis, the material in the "dirty" containers may be discarded. The "clean" containers may be moved up to the dirty position, and then fresh fluid added to clean position. DI water may be drawn from the large system DI water container, and discarded after each use. Target retrieval solution may be drawn from dedicated containers, and may be recycled or discarded after each use.

Returning to the aspect of monitoring or capturing information, an embodiment of the system may be designed to monitor replenishable supply information, such as the status of buffers, reagents, stains or the like. By monitoring for a potential need for replenishable supplies, the system may not only provide the replenishable supply information monitor 403 shown in FIG. 22, but it may also relieve operators of some concerns. It may also remove at least one possibility for human error. Significantly, the system may also act to automatically notify any number of people relative to the information monitored. With respect to replenishable supply information, the system may notify a user, an operator, an administrator, or even a supplier of an actual, potential, or impending need to replenish supplies. As such the system may be considered as including an automatic notice element 404, or an automatic operator replenishable supply notice element, an automatic supplier replenishable supply notice element, or the like.

In a similar fashion, an embodiment of the system may monitor or capture information that is of interest to the continued or continuous operation of the device. As such, it may be monitoring instrument maintenance information. This may include, but is not limited to monitoring part cycle information, ranging from a gross information such as age of the device, estimated number of cycles, to even monitoring specific information such as monitoring individual part cycle information (e.g., how many times and actual valve was turned on or off, etc). By including (see FIG. 22) an instrument maintenance monitor, an instrument maintenance information monitor 405, a part cycle monitor, or an individual part cycle monitor 406, the system may facilitate not only enhanced reliability and continuous operation, but it may permit preventative maintenance such as maintenance based on product cycles or mean times between failures. Naturally, it may also use the automatic notice element 404 such as providing an automatic maintenance notice element to inform a wide range of persons of such issues. Of course, a large variety of information may be monitored; embodiments of the system may monitor or capture information that relates to material requirements, such as expiration dates, lot information or the like. Thus the present invention may include a material requirement information monitor 407 (FIG. 22) so that it acts to automatically monitor material requirement information. This may be a product expiration information monitor 408 that may even act with respect to an upcoming expiration and may even cause the set of automatically advance notifying a person by providing an automatic advance expiration notice element. For items that may be very important, there may even be multiple notices either concurrently or sequentially and as such the system may include a multiple advance expiration notice element. Another type of information that may be monitored is historical usage information such as information of a statistical or past nature. Thus the system may include an historical usage information monitor 409 (FIG. 22). From this, predictive estimates may even be made such as a likely date upon which to order an item or the like. Through monitoring predictive usage information, this may be one way the system may be able to provide an automatic predictive need notice element or even a predictive usage information element 410 (FIG. 22). The system may also provide for a user statistical information monitor so that it can assemble and monitoring user statistical information and act on this such as by comparing to other historical or statistical information or the like. The present invention may also be configured to monitor sample process efficacy information such as by assuring particular protocols are followed or the like and may thus provide a process efficacy information monitor 411 (FIG. 22). Monitored information may be extrapolated to permit a totalization capability 413 (FIG. 22) by adding up individual usages to know amounts left or otherwise impacted by operation. This may include totalizing usage information for an item such as a reagent or an individual part's cycles. Such a capability may serve as a totalization usage information monitor, a reagent totalization, or a part cycle totalization. The system may also report cost per test and other such synoptic information that may be important to the economics and efficiency of instrument operation from a practical perspective. By having a data capture element 414 (FIG. 22), the system may generate data that may include or permit analysis or use of a variety of aspects, including but not limited to: number of occurrence data, part operation data, amount of usage data, and amount of material used data. Such data may, of course, have a like element, perhaps a subroutine, to do or generate the various function or data involved.

In some embodiments, an imaging device such as an image-capture 2-D optical sensor, perhaps a CCD camera, or the like, may be used to determine the position of the sample on the slide, providing for greater accuracy during sample processing. Embodiments of the sample processing system 101 may further provide sample diagnostic capabilities. Accordingly, in some embodiments, a device may analyze samples. A camera may be used for diagnostic purposes. In some embodiments, the sample may be scanned for further analysis, potentially by computer. The camera can also be used 1) as an area locator, 2) to locate a tissue area, 3) to apply reagent based on location and area. The scanned image may be analyzed for reagent analysis or other analyses.

The system may also generate or also monitor subject sample data. Relative to the imaging device, the system may monitor or perhaps capture image data, such as sample image data, substance image data, system image data, and even pre- and post-event image data. Each of these may be systematically stored for some purpose. Each of these may correspondingly be considered to present an appropriate element such as a system image data capture element, a substance image data capture element, a sample image data capture element, and a pre- and post-event image data capture element. In addition, there may be included a multiple image data capture element so that more than one image may exist to prove or evidence an aspect of the processing. Again, the system may act to systematically store and one or the multiple images so created. Collections of like data, such as groupings of individual sample process data, individual slide log data, and even type of protocol data may also be created.

The processing of samples may be accomplished according to some preferred embodiments as shown in FIG. 19 and FIG. 20 consistent with features of the present invention. Variants of these protocols and processing steps, or other processing steps, may be accomplished consistent with the present invention. One processing sequence may broadly comprise the pre-processing of a sample, if needed, such as de-paraffinization (as previously described), and further comprise target or epitope retrieval (as previously described), and sample staining. In some embodiments, specifics of in-situ hybridization (ISH) may be addressed. Embodiments of ISH may require a small volume of reagent, such as 15 micro¬ liters, to be placed on the sample. Heat control may be maintained between about 95-100 C and kept constant for a period of time. Temperature may then be lowered in a controlled manner. Furthermore, fluorescent staining or tagging in IHC or ISH (FISH) may be performed consistent with the features of the present invention.

As mentioned, the sample processing system may automate the processing of samples mounted on carriers or slides. This configuration of the system allows for the flexibility for both continuous, individual, and batch processing of slides with the design lending itself to meet established laboratory workflow demands. The multiple independent and redundant slide processing subsystems found within the system may also maintain its ability to process each slide independently.

The automatic processing may be achieved by designing a system with automated process operation capability or sequencing through at least some steps without human intervention. This may be controlled by or act in response to a process operation control system 171 (FIG. 22). This may be provided through hardware, software, or some combination of the two. FIG. 22 provides one conceptual embodiment that depicts some of the various capabilities. Of course, the user needs the ability to specify the nature and sequence of the various steps or acts desired or even the appropriate priority or other scheduling parameters to be used. This can be accomplished by an input parameter capability 173 through the inclusion of even a sample process parameter input. Input can be retained by the creation of stored parameter process data 174 so that the system can achieve the aggregate or perhaps plurality of process operations desired and thus the input may be an aggregated sample process input. In order to facilitate uninterrupted processing, the input parameter capability 173 may be configured as an independent process parameter input with respect to the process operation control system 171, such that acts caused by the process operation control system 171 are unaffected by any action with respect to the independent process parameter input. Further, the input parameter capability 173 may also be configured as an autonomous input functionality through the inclusion of an autonomous input element.

With the desired types of processing input, the system may act to automatically schedule the various events perhaps through a schedule element 605. These events may be considered as making up an aggregated event topology in that there is an aggregation of desired events and in that the events themselves present some topology or contour for a processing sequence. This topology may include only the events or it may include certain goals such as a particular prioritization or outcome desired. When using an initial input, the system may achieve scheduling of the events in the manner desired. Of practical importance may be the ability of an embodiment of the invention to permit and facilitate operator changes to the initial aggregated event topology. Significantly, these changes may be achieved dynamically, such as while other parts of the system are continuing processing. In facilitating changes while otherwise operating with little or no interruption, the system may act to achieve adaptive scheduling. This may begin as adaptive scheduling of an initial aggregated event topology and may evolve into adaptive scheduling of an altered aggregated event topology. This may occur on individual or perhaps stand alone devices, such as a stand alone stainer, or it may occur on an inter machine basis, such as by using an inter machine schedule indicium or an inter machine schedule element. Regardless, it should be understood that the scheduling of an altered topology may occur after commencing an initial automatic processing routine.

The alteration of the aggregated event topology may include any variety of actions that effectively alter an initial setup. These may include but are not limited to: altering the aggregate, such as perhaps adding a sample, deleting a sample, changing a sample, or altering the topology such as accepting a user change input such as merely a change in priority. They may also include accepting a temporary user change such a change that a user wants to see the effect of but may not wish to implement. Thus the system may include a sample addition element, a sample deletion element, more generally a sample change element 601 (FIG. 22), or a temporary user change element, each of which may be considered as creating some type of altered aggregated event topology. To permit a user decision, embodiments may include functionality or subroutines for activating a user change or undoing a user change. These may be considered a user change activation element or a user change undo element. Such selection may be presented in conjunction with a results display element 602 of some sort such as an effect synopsis display element, a temporal impact display element (e.g., the time impact on one or more samples to be processed as a result of the alteration), and even an estimated temporal impact display element, whereby the time effect is only estimated.

As a result of some type of alteration in the aggregated event topology, the system may reschedule events. This rescheduled sequence may be used to interrupt or may provide an interrupt 603 (FIG. 22) relative to the initial sequence and to thereafter continue revised automatic processing according to the altered aggregated event topology. As can be understood, this may be accomplished without completing the initial automatic processing. The rescheduling may be programmed to achieve a variety of results and then to compare with its "best" depending on how the operator or system define that goal. Achieving a variety of results can be accomplished by simulating runs or perhaps a portion of a run and comparing the results of that simulation. The simulation may be of varied sequences set up according to certain parameters as explained below. By so doing, embodiments may include varied-parameter robotic control simulation functionalities 606 (FIG. 22), that is programming that simulates robotic operations based on differing parameters. These varied- parameter robotic control simulation functionalities 606 may be responsive to the aggregated sample process input by acting on the data the input creates. Specifically, the system may run multiple simulations for the same aggregated event topology with each simulation using different criteria to determine the sequence of steps. The results of these simulations may be indicium that can be used and compared. Comparison may be achieved by an automated process simulator comparator 604 (FIG. 22) which may look at any indicium resulting from the particular simulation being considered. From the indicium, a decision may be made and a particular set of parameters may be determined to cause an enhanced, if not optimum, sequence for a desired goal. These parameters may then be used in a preferred functionality robotic control generator 607 which may then actually create the sequence that is used for the desired process operation. In this fashion, the system may have a process generator that is responsive to the automated process simulator comparator and from which an automated process functionality may be created.

As mentioned, the simulations may take into consideration a variety of input for factors, including a user parameter input. Of course, there are a variety of parameters that may be considered as the rescheduled sequence is determined perhaps by comparing indicium (e.g., any value having information relative to that particular model) relative to a particular model. These may include but are not limited to: a substance priority parameter, a reagent grouping parameter, a robotic movement parameter, a sample location priority parameter, a sample proximity priority parameter, a sample insert time priority parameter, a user input parameter, a user priority parameter, a sample time since last processing priority parameter, a time-based priority value parameter, and a sample weighting parameter.

The system may compare the results, perhaps by software that may act as a comparator 604 (FIG.22) . The elements compared may be elements such as comparing processing time indicium, comparing completion time estimates, comparing substance cost estimates, or comparing sample priority assignments, and as such may be considered as having a robotic control simulation results comparator, a sample time since last processing priority parameter robotic control simulation functionality, a time-based priority value parameter robotic control simulation functionality, a substance priority parameter robotic control simulation functionality, a completion time estimate comparator, a substance cost estimate comparator, a sample priority assignment comparator, a repetitive process simulator comparator, and even a qualitative analysis comparator. As mentioned earlier, to facilitate some type of comparison., it may use indicium, such as an initial robotic control indicium and a second robotic control indicium.

In establishing a system that is practical, it may be advantageous to include — at least initially for calculations time concerns — a limited number of different simulations. For example, two or three may be included and may thus be considered a first control simulation functionality, a second control simulation functionality, and a third control simulation functionality. By establishing a system with a sample time since last processing priority parameter robotic control simulation functionality the system may assign a higher priority to samples that have not had any or perhaps particularly important activities for some time. By establishing a system with a robotic movement parameter robotic control simulation functionality it may take into consideration how far a robot needs to move to assign priority to items that require less movement. By establishing a system with a substance priority parameter robotic control simulation functionality, it may include consideration of the fact that some substances are particularly concerning either because of cost, rinse needs, toxicity, or the like. Finally, in making a comparison to determine which parameters yield a more desirable sequence, the system may include an enhanced temporal scheduler element so that the system automatically evaluates which parameters are likely to yield the fastest processing time. Naturally, this enhanced temporal scheduler element may be based on a total-sample basis or may be based on some subset thereof. It may even be based on individual samples such as for a stat run or the like. Thus the robotic control simulation results comparator 604 may act to provide an enhanced rescheduling of an altered aggregate event topology. In implementing the revised sequence, the system may provide a seamless initial adaptive schedule functionality interrupt and may act to seamlessly, perhaps without perceptible discontinuity, interrupt the initial sequence and continue with the new one. Further, since the simulations may be time consuming, it is possible do only an initial comparison, perhaps such as merely comparing two differing functionalities, to then select one of them such as an initially preferred robotic control functionality and to then continue more simulations and comparisons. From this continued effort, there may be discovered an even better set of parameters and thus the system may thereafter implement a second preferred robotic control functionality as perhaps a better solution. Naturally, continued simulations and comparisons may occur.

As may be understood by the above, rescheduling due to an altered aggregate event topology may be impacted by a number of factors. As but one example it may be understood in shortening time for overall processing, the location of a particular substance or a particular sample may be important; the longer distances between samples or substances, the slower the processing. Because of this type of factor, it is possible that the system may actually consider, simulate or otherwise assess factors and may suggest actions that may yield desired results. For example, the system may display at least one suggested sample location, a suggested sample drawer location, a suggested stainer location, or the like. From this, the user may be able to accept a proposed action and may even be able to accept or reject the suggestion. Thus the system may display a suggested user selection. This may even be the act of displaying a temporally enhanced suggested user selection through providing a user selection menu or the like. From this, the system may accept a user parameter input through a user selection menu. The results may even be summarized to display a synopsis of the effect due to the alteration, such as to display a temporal impact due to the alteration. Naturally, this may be estimated and the system may act to display an estimated temporal impact. Whether impact based or suggestion based, the system may provide the user valuable input and in this manner it may actually provide a suggested sample location element, a suggested sample drawer location element, a suggested stainer location element, a suggested user selection element, a temporally enhanced suggested user selection element, or the like. Naturally, such activities as well as any rescheduling or simulating may be the result of an operator request, the system sensing an operator access event, the system accepting a user change, or even some type of operator access event sensor, such as a drawer sensor or the like.

Similar to the act of suggesting to the operator a particular action that may enhance scheduling, the system may act to inform the operator of needed events or the like. If a particular substance is required but is not present in the machine (likely as sensed by the device itself perhaps through the optical sensor), the system may automatically prompt an operator for a particular action needed, such as inserting the needed reagent or the like. In downtime or otherwise, the system may even repetitively automatically check if an operator action is needed. As such the system may include an automatic operator need prompt 608 (FIG. 22). It may also provide a variety of information such as real time status information, pending sample information, a real time completion estimate for an aspect (e.g., a sample, a drawer, a batch, or the like). Each of these may be accomplished by software and hardware perhaps by including a real time status information element, a pending sample information element, or a real time completion estimate element, each shown conceptually as the information element 609. As to any of the above capabilities, such may not only act independent of the automated process operation capabilities, but where applicable, they may be fully functional even without the presence or operability of the automated process operation capability (which itself may or may not be in a process device). They may be achieved in a variety of manners, including by providing a separate full function computer 181 (e.g., separate from the capability provided or required by a process system) or that may be programmed to accomplish the desired function. In addition, in order to accomplish a goal of addressing practical and institutional needs, any capability may be configured to provide simplified use and may even be available in a highly simplified level of detail. This may be a "wizard" type of system where there is a "step-by-step" method for functions such as adding slides, achieving the desired input, or the like. Such an aspect may even be simple, regimented, and somewhat inflexible. A structured or simplified input can facilitate input by persons not required to have the full spectrum of skills necessary to be responsible for the operation of the sample processing system 101.

As part of the functions of monitoring or perhaps allowing play back of events, the system may include some type of data capture element 414 (FIG. 22). As may be appreciated from the initial discussion of the types of actions potentially needing to be programmed, the data capture element 414 may capture individual movement data, only robotic action data, individual robotic movement data, individual operation data, or even individual usage data. Thus the data capture element 414 may be an individual movement data capture element, a robotic action data capture element, an individual robotic movement data capture element, or an individual operation data capture element. All or any part of this data may be systematically stored such as storing all important details, only particularly important details (e.g., relative to highly sensitive valves, substances, or the like) or e ven only a significant number of details relative to sample process operations. Thus the data capture element 414 may be a systematic process detail capture element. Once captered, this data may be stored in a number of fashions. There may be a memory location at which such data resides and this may thus represent a significant process detail memory 412. It may also represent a subject sample data capture element and any of the memory types mentioned earlier may be used for such a purpose. In storing the data, the system may create a segmented computer file, that is a file that contains only such data so that it is not as easily manipulated as other files. This may aid in assuring the accuracy or even certifiability of the events depicted. For instance for any particular sample, there may be automatically generated upon request a simulation ~ perhaps with a time base appended ~ of what happened to that particular sample as well as pictures of the sample before and after its processing. The data so stored may even be created as an inalterable computer record and perhaps may even include an integral change indicia that can prove its accuracy. When stored, the system may create a common format computer record so that user can easily work with it or it may create a proprietary format computer record that cannot be altered or the like. Thus the significant process detail memory 412 may represent a segmented computer file memory element, an inalterable computer record memory element, an integral change indicia memory element, a common format computer record memory element, or a proprietary format computer record memory element.

The capture of data may include time of occurrence data, such as actual date data, actual time data (e.g., UTC, etc.), precise time data (e.g., hours, minutes, seconds), relative time data, absolute time data, initiation time data, and even completion time data (e.g., process, protocol, motor operation events, or the like). Again, the data capture element 414 may include, but is not limited to, a time of occurrence data capture element, an actual date data capture element, an actual time data capture element, a precise time data capture element, a relative time data capture element, an absolute time data capture element, an initiation time data capture element, or a completion time data capture element.

One item that may be of particular user desire is the fact that the data capture element

414 may represent an individual sample process data capture element, an individual slide log data capture element, a type of protocol data capture element, and even an individual slide log data capture element. There may also be a real time individual slide log data display to show actual processing as it occurs. As used above, the slide identification information may represent any information unique to a particular slide, such as a serial number, patient number, patient name, unique image, or the like. In keeping with privacy concerns, there may also be coded or perhaps encrypted identification information or internal identification information that others cannot use to identify the particular patient involved or the like. As discussed below and as shown in FIGS. 8 & 9, the overall system may include a number of staining instruments and thus the input can include preferred stainer information (which may or may not be indicated or accepted by the automated system). Provision can also be included to achieve a rush test and as such there may be an immediate, urgent, or otherwise known as stat (an often used medical term for immediate) process request information element. Such may also be linked with user privileges information so that only certain individuals may displace other tests to create a different priority. Of course all permutations and combinations of the above may be included.

For automated operation, the input may create data such as parameter process data

174 that may be stored at some location. To provide autonomous operation, it may be independently stored perhaps in a physically independent memory even at a location remote from an actual stainer itself. This may be accomplished by utilizing a primary or secondary storage perhaps of a separate full function computer programmed or configured to accept and/or store data. In such a fashion, the computer may contain what could be considered as an independent process parameter memory 174. Since the computer is likely physically separate, it may be considered to have a physically independent memory perhaps even a remote location memory if it is remote from the process equipment.

By using independent memory and independent other functionality, the system may facilitate full operational functionality of the automated process operation capability. Since the automated process operation capability is fully operational during operation of either the memory or input, the storing or inputting or other function can be conducted without interrupting the process operation. Thus the inputs can be later accessed at a process time independent of the time of accomplishing slide process parameter input or storing. In addition, entry or storing may also be accomplished at least in part concurrently with the processing of certain samples. This processing may even be initiated significantly after completion of the slide process parameter input action. Such may occur at least about one hour after the input, at least about three hours after the input, at least about eight hours after the input, at least about one day after the input, at least about two days after the input, and at least about one week after the input.

As mentioned briefly above, once the information is either monitored or captured, the present invention may act to automatically inform at least one person who may find the information useful. The automatic notice element 404 mentioned earlier may be configured to act as an automatic exteriorly-consequential information notice element by relating largely to that type of information. Of course, the automatic notice element 404 (FIG. 22) may act in response to the step of monitoring the particular information involved. For example, if it is monitoring operationally-altered outside information, the automatic notice element 404 may act as an automatic operationally-altered outside information notice element. For process events that are merely captured and not automatically monitored, a person may prompt the system upon which it may provide information by some type of display 415. This display (in its broadest sense) may reveal at least some information, perhaps relative to sample process operations to at least one person. If the display reveals significant process detail information, it may be considered as a significant process detail information display. Further if it displays at a separate location there may even be a significant process data transfer element to facilitate remotely displaying such information. As such the display 415 may be considered a remote process detail information display. As mentioned earlier, the system may provide for a real time information display, that is a display that reveals information at about the time it occurs. By real time displaying information remotely, the operator or any other interested person may be able to "watch" or monitor the progress of the instrument from another location — perhaps even the other side of the world. This may be particularly valuable when there is a real time display of individual slide log data as mentioned above.

One type of display 415 (FIG. 22) that may be noteworthy is the fact that embodiments of the invention may create a simulated motion display. The simulation may visually show an element moving on a screen just as the robot head actually moved when it operated. Embodiments can provide sequential playback capability so that one could also "watch" the instrument just as it operated at some earlier time. There may also be an altered speed sequential playback capability, a user alterable speed sequential playback capability, or merely a high speed sequential playback capability perhaps all with or without pause or slow motion capability. With this capability, the display 415 may represent a simulated motion process detail information display. The system may thus include a sequential playback element, an altered speed sequential playback element, a user alterable speed sequential playback element, and a high-speed sequential playback element.

All this information must, of course be used by some person. Any interested person may have the information available to them, such as an operator (e.g., anyone responsible for all or a portion of a process or the instrument), an instrument operator (e.g., an individual physically responsible for all or a portion of a process), an administrator (e.g., a person managing operators or perhaps responsible for order placement), a substance or other supplier, or even a manufacturer, such as for support and maintenance capability. For events that may require external actions (e.g., ordering more reagent or the like), the system may automatically notify at least one of these types of people and thus the automatic notice element 404 (such as a display which may be visual or otherwise) may be considered as representing an automatic operator notice element, an automatic administrator notice element, an automatic supplier notice element, or an automatic manufacturer notice element. It may also be considered as representing an automatic operator exteriorly-consequential information notice element, an automatic administrator exteriorly-consequential information notice element, an automatic supplier exteriorly-consequential information notice element, or an automatic manufacturer exteriorly-consequential information notice element.

Notice may be given at a variety of times. The system may act to automatically advance notify a person such as of an upcoming expiration date or of a need to reorder in advance. In so doing it may have or have input to it some type of lead time information that tells it how early to take the action. By properly configuring a lead time information data element 416 (FIG. 22), lead time may vary by location and situation, for example a machine around the world or used continuously for critical processing may have a longer lead time than a machine right next to a supplier or used only sporadically. Order lead time information, reagent order lead time information, maintenance lead time information (any of which may vary over the course of a year or from time to time) may be utilized and as such the lead time information data element 416 may represent an order lead time information data element, a reagent order lead time information data element, or a maintenance lead time information data element.

Notice itself may be displayed in a variety of ways. The system may automatically E- mail a person through inclusion of an E-mail notice element; it may automatically print out (including faxing) a notice by having an automatic printout notice element. Among other possibilities, it may automatically utilize a telephone line for simulated or reproduced voice or other information by having an automatic telephone line utilization element.

The actual event of providing notice may be automatic or it may by caused by some type of user prompt 417 (FIG. 22). By accepting a monitored information user prompt the system may represent a monitored information user prompt. The prompt itself may be a mere software selection or even a mere click-on items such as a software displayed button or the like. Whether displayed and acted upon remotely or at the actual robot-containing housing, such a user prompt 417 may cause a remote access connection to be established and as a result at least some significant process data may be displayed. In such a manner the user prompt may represent an information access prompt element, a software selection element, or a remote access element.

In some embodiments, the system may be comprised of independent or perhaps redundant slide staining modules (some embodiments may comprise eight modules) as shown for some embodiments in FIGS. 15 and 16. Throughput may be based on the time to first result with the system allowing access to completed slides as soon as a staining module has completed the scheduled staining tasks. The multiple independent or redundant staining modules may allow for both continuous and batch processing of slides. Additionally, each independent staining module may also allow for the independent pre-treatment and staining of each slide. A carrier retaining assembly, such as a slide retaining assembly, may be used to introduce slides to be processed into the drawer 110, the drawer, slide retaining assembly, and components thereof forming a stain module. The slides may occupy one or more positions of the slide retaining assembly, such as at carrier retention devices, up to the capacity of the slide retaining assembly with the potential for each slide being processed independently of other slides configured with the slide rack. Embodiments of the stain modules, drawers, slide racks, and components thereof are also shown in FIG. 16. FIG. 16 also provides other embodiments of system features, such as an embodiment of the arm 120 and the component features of the arm.

Slide retaining assemblies having one or more slides and even reagent containers may be introduced into the staining or reagent modules by introduction into drawers 110 (FIG. 15)one at a time or in any combination until all or an appropriate number of staining modules are appropriately occupied. There may be no restrictions as to the order, number or timing of when the slide retaining assemblies are introduced into the system, the system may also allow for adaptive scheduling of sample loading. Staining modules, and in some embodiments the drawers of the staining modules, may lock out access to the slides during the processing period and may release them to the operator upon completion of the staining or other process on the last slide of that module. In some embodiments, the order in which the slide retaining assemblies are released may be dependant on the time required to process the last slide of the retaining assembly. Slides may even be processed in the most time efficient manner independently of the order to which they were introduced into the system. The system may provide an optimum or merely an enhanced temporal scheduling of the various sample process steps. To accomplish this, the system may automatically schedule steps that are interspersed for an enhanced time result. This interspersing may be an interleaving of a number of process operations and even an interleaving of a number of individual sample operations. In addition to interleaving steps, the system may sequence the individual sample operations. Regardless as to how programmed, it may be configured through hardware or software or a combination of each to provide an enhanced temporal scheduler element 179 (FIG. 22), a process operations interleave element, an individual sample operations interleave element, or even an individual sample operations sequence element. These can be created by integrating the automated process operation capability and either the parameter data or perhaps some replicated portion of that parameter process data (as mentioned later) and can thus act to create an interspersial robotic control functionality 175.

Slide retaining assemblies having one or more slides may be introduced into the staining modules by introduction into drawers 110 (FIG. 15) one at a time or in any combination until all staining modules are occupied. There may be no restrictions as to the order, number or timing of when the slide retaining assemblies are introduced into the system, the system allowing for adaptive scheduling of sample loading. Staining modules, and in some embodiments the drawers of the staining modules, will lock out access to the slides during the processing period and may release them to the operator upon completion of the staining process on the last slide. In some embodiments, the order in which the slide retaining assemblies are released is dependant on the time required to process the last slide of the retaining assembly. Slides may be processed in the most time efficient manner independently of the order to which they were introduced into the system.

The control of the processing samples may be accomplished according to the following preferred embodiments, one preferred embodiment 184 is shown in FIG. 23, although other processing may be accomplished consistent with the present invention.

Control of the sample processing may be accomplished by a dynamic scheduling algorithm, and in preferred embodiments, in accordance with the continuous or batch processing previously described. The processing sequence may be controlled, in preferred embodiments, such that the various steps of a protocol for samples may be automated by one or more algorithmic controls. A preferred control may be accomplished as follows: 1) selecting a first protocol step, 2) selecting a second protocol from a restricted list of menu items that are compatible with the first protocol step, and 3) selecting subsequent protocol steps from a restricted list of menu items that are compatible with the preceding protocol step.

In expanded systems, a sample processing system manager, such as a computer server may be connected with a number of individual sample processing systems. These may represent automated slide stainers 101 or even stand alone automated slide processing system such that they are fully capable of functioning with connection to other devices. In systems where a connection does exist, the capability of electronically connecting a number of automated slide stainers or automated sample processing systems or label printers 200 (FIG. 23), may be provided. As mentioned earlier, there may be one or more separate full function computers 181 connected. These may be connected through a hub 193. There may be a multitasked central processing unit resource on either the stainer or the computer or there may be a number of central processing units that are configured to avoid using or implementing a multitasked central processing unit resource relative to the process operations in order to maintain full independence or perhaps even autonomous operation. The connection, whether for input or other operation may also be a remote link (including ability to be made remote such as in detachable memory) such as an internet connection element, a telephone line connection element, a wireless communication element, or even a detachable memory element. In a preferred embodiment, connection among perhaps a number of process systems and perhaps a number of computers, such as workstations and a server (the latter residing either separately or as part of a workstation), may be achieved by use of a local area network (LAN), such as a group of computers and associated devices that share a common communications line or perhaps wireless link and may even share the resources of a single processor, memory, or server within a small geographic area (for example, within an office building or complex). A local area network for this type of system may also include features such as but not limited to: an Ethernet element, a token ring element, an arcnet element, a fiber distributed data interface element, an industry specification protocol, a bluetooth-based. element, a telecommunications industry specification using a frequency band of 2.45 GHz, a communication specification applying an IEEE 802 standard, a frequency hop communication specification, a shared common link element, a transmission control protocol/internet protocol communication element, a packetized information protocol, a shared protocol, a proprietary protocol, and even a layered protocol exchange system. By providing an electronic connection 176 between various resources, the local area network such as the stainer network 184 (a network dedicated to only the stainer or perhaps sample processing resources for integrity, security, and other purposes) in one embodiment may transmit a electronic memory address to achieve access to the appropriate information. Connection may also be established to a lab network, facilities intranet system, or even a lab information system 195 such as thorough a bridge 194 (FIG. 24).

As mentioned, connection may be accomplished over internet connections but more preferably is accomplished over local area network connections . Each sample processing system may be individually controlled, in some embodiments, by a PC attached with, internal to, or otherwise provided. Data sharing between sample processing systems and the system manager may be performed to allow identification, tracking, and. status of sample batches, reagents, and other agents and components of the sample processing system. A determination of which system has which reagents, reagent type, slides and protocols may be performed. Log files for each processing sequence, protocol, ox slide can be generated for monitoring processing status. Database maintenance (including but not limited to purge, compact, back-up, database/list, and archive functions) and system diagnostics (including but not limited to exercising active system components to verify proper operation and assisting in troubleshooting efforts) may be accomplished manually or automatically.

The system may be configured to automatically access the required data through operation of the process operation control system 171 (FIG. 22) by inclusion of an automatic memory access element. This access may be achieved by specifying an electronic memory address that may be transmitted by a electronic memory address element 178 perhaps over a local area network and may be followed by automatically replicating that data on some a memory aspect appropriate for operation such as an automatic data replication memory. This memory may include but not be limited to: a volatile memory functionality as implemented by a volatile memory element, a random access memory functionality as implemented by a random access memory element, a non-volatile memory functionality as implemented by a non- volatile memory element, an electrically erasable programmable read only memory functionality as implemented by an electrically erasable programmable read only memory element, a main storage functionality as implemented by a main storage element, a secondary storage functionality as implemented by a secondary storage element, a cache memory functionality as implemented by a cache memory element, and even a detachable memory functionality as implemented by a detachable memory element.

A control interface may be provided for the operator, such as a graphical user interface (GUI), and may accommodate various languages. Help menus may be provided to assist in sample processing. Password protection features can be provided and even administrator control over at least some aspects. This may include the capability to include administrator limitations on the functional availability of any aspect of the system or of specific stainer availability or functionality, certain reagent availability functionality, certain protocol availability functionality, patient identification information access functionality, process priority request functionality, and immediate, urgent, or stat process request functionality. By including an administrator control element 180, the system may have an administrator-implemented user limitation element, a specific stainer availability limitation element, a certain reagent availability limitation element, a certain protocol availability limitation element, a patient identification information access limitation element, a process priority request limitation element, an immediate, urgent, or perhaps stat process request limitation element, a user privileges input element, and even a user group privileges configuration or input element.

Control of the sample processing may be accomplished by a dynamic scheduling algorithm, and in some embodiments, in accordance with continuous, or batch processing previously described. The processing sequence may be controlled, in preferred embodiments, such that the various steps of a protocol for samples may be automated by one or more algorithmic controls. As part of input to establish the desired control functionality, user or other input may be accommodated as follows: 1) selecting a first protocol step, 2) selecting a second protocol from a restricted list of menu items that are compatible with the first protocol step, and 3) selecting subsequent protocol steps from a restricted list of menu items that are compatible with the preceding protocol step.

After all data is input, the system may act to determine operational readiness by inclusion of an operational readiness determination element 177 that may be programmed to assess if appropriate resources, drawers, slides, reagents, or other aspects are present or available to the system. As mentioned above it may notify an operator of a need if any exists. Once an appropriate operational readiness is determined, the system may prompt initiation of access of the input data to electronically determine operational availability of a variety of items. These may include but are not limited to: an individual sample element through inclusion of an individual sample readiness determination element, a defined group of samples through inclusion of a defined group of samples readiness determination element, a physically grouped collection of samples through inclusion of a physically grouped collection of samples readiness determination element, a slide drawer component through inclusion of a slide drawer component readiness determination element, a stand alone automated slide processing system through inclusion of an stand alone automated slide processing system readiness determination element, a slide stainer system element through inclusion of a slide stainer system readiness determination element, and even a user initiated prompt signal such as might occur to force or activate the system manually by the inclusion of a user initiated prompt signal determination element.

There may even be timing tolerances, referred to in some embodiments a.s "bubble tolerance", that may be controlled as between steps, such as between aspiration cycles. Additional control may be accomplished through timing algorithms to determine time tolerances of components of the processing system, such as the monitoring of "shelf life" or viability of reagents. Furthermore, adaptive scheduling of sample and slide insertion and removal into the system, as previously described, may be accommodated on an on-going basis throughout operation of the sample processing system.

As can be easily understood from the foregoing, the basic concepts of the present invention may be embodied in a variety of ways. It involves both sample processing techniques as well as various systems, assemblies, and devices to accomplish sample processing, input, and other functions, hi this application, the sample processing techniques are also disclosed as part of the results shown to be achieved by the various systems, assemblies, and devices described and as steps, which are inherent to utilization. They should be understood to be the natural result of utilizing the devices as intended, and described. In addition, while some devices are disclosed, it should be understood that these not only accomplish certain methods but also can be varied in a number of ways. Importantly, as to all of the foregoing, all of these facets should be understood to be encompassed by this disclosure.

The discussion included in this application is intended to serve as a basic description. The reader should be aware that the specific discussion may not explicitly describe all embodiments possible; many alternatives are implicit. It also may not fully explain the generic nature of the invention and may not explicitly show how each feature or element can actually be representative of a broader function or of a great variety of alternative or equivalent elements. Again, these are implicitly included in this disclosure. Where the invention is described in device-oriented terminology, each element of the device implicitly performs a function. Importantly, neither the description nor the terminology is intended to limit the scope of the claims which may be included at any time.

It should also be understood that a variety of changes may be made without departing from the essence of the invention. Such changes are also implicitly included in the description. They still fall within the scope of this invention. A broad disclosure encompassing both the explicit embodiment(s) shown, the great variety of implicit alternative embodiments, and the broad methods or processes and the like are encompassed by this disclosure and may be relied upon at any time.

Further, each of the various elements of the invention and claims may also be achieved in a variety of manners. This disclosure should be understood to encompass each such variation, be it a variation of an embodiment of any apparatus embodiment, a method or process embodiment, or even merely a variation of any element of these. Particularly, it should be understood that as the disclosure relates to elements of the invention, the words for each element may be expressed by equivalent apparatus terms or method terms — even if only the function or result is the same. Such equivalent, broader, or even more generic terms should be considered to be encompassed in the description of each element or action. Such terms can be substituted where desired to make explicit the implicitly broad coverage to which this invention is entitled. As but one example, it should be understood that all actions may be expressed as a means for taking that action or as an element which causes that action. Similarly, each physical element disclosed should be understood to encompass a disclosure of the action which that physical element facilitates. Regarding this last aspect, as but one example, the disclosure of a "retention element" should be understood to encompass disclosure of the act of "retaining" ~ whether explicitly discussed or not - and, conversely, were there effectively disclosure of the act of "retaining", such a disclosure should be understood to encompass disclosure of a "retention element" and even a "means for retaining".

Any patents, patent applications, publications, or other references mentioned in this application for patent are hereby incorporated by reference. In addition, as to each term used it should be understood that unless its utilization in this application is inconsistent with such interpretation, common dictionary definitions should be understood as incorporated for each term and all definitions, alternative terms, and synonyms such as contained in the Random House Webster's Unabridged Dictionary, second edition are hereby incorporated by reference as well as the definitions presented by searchStorage.com, such to be considered as representing the meaning of the terms as understood by computer professionals. Finally, any priority case for this application is hereby appended and hereby incorporated by reference.

APPENDIX:

Reduction of the primary antibody incubation time with 50% Aim To verify that the SAW chips integrated on the AS match the performance previously observed with the manual IHC experiments. The goal is to show that SAW micro agitation can compensate for a 50% reduction of the incubation time of the primary antibody.

Method CK NV test protocol (AEI/AE3 1:900, target retrieval in S2367, K5007) used on the AS. All drop zones tested (three sections on each slide) using sections of screened tonsil and multiblock placed in each drop zone just above the SAW chips.

The SAW units will be turned on only during the 15 minutes of antibody incubation and these will be compared to control sections on an additional AS rack incubated with primary antibody for 15 and 30 minutes.

Two other antibodies (A0452, 1 :100) and Ki67 (Mib-1 , 1 :400) were also tested accordingly.

Results Generally, none of the slides exposed to SAW mixing, irrespective of chip-design, scored less than control slides incubated for 30 minutes with the CK antibody. This meets the first object and we conclude that the initial results observed can be reproduced on the SAW-AS rack from Advalytix.

Figure A1 , page 66 and Figure A2, page 66a shows the results of the first testing series. Figure A1 : CK AE1/AE3 staining of slides using S1700 and K5007. SAW on during 15 minutes of primary antibody incubation. Slide score (1-4) is relative (±) to control slide that have been incubated 30 minutes without SAW (slide # 15 scored to be 2 used). 3/8-05 Large tonsil placed above each SAW chip (tissue very inhomogeneous and scoring was hindered). H 143 Tonsils placed above middle SAW unit and between the lower and middle unit (scored together). H 144 Tonsils placed above middle SAW unit and between the middle and upper unit (scored together) Figure A2: CK AE1/AE3 staining of slides using S1700 and K5007. SAW on during 15 minutes of primary antibody incubation. Slide score (1-4) is relative (±) to control slide that have been incubated 30 minutes without SAW. H145 Large multiblock placed above middle SAW unit. H146 Small Multiblock placed above SAW units. H147 Small Multiblock placed between SAW units.

Figure A1 and A2: The results of three individual experiments, with a reduced incubation time for the primary antibody. The different configurations of the SAW slides (slide 1-12) are indicated by color and the Power (dBm) used on the chips is also shown. A reference slide incubated only 15 minutes without SAW (Ref 15 min.) is shown as well as two control slides incubated for 30 minutes without SAW. For the control slides the actual IHC score is shown and for the rest of the slides the difference between that slide and the rest of the slides are given.

From Figure A1 and Figure A2 (see also FIG. 26) it is clear that we are able to reduce the CK incubation time with SAW micro agitation to 50% and still get comparable stainings using both tonsil and multiblock tissue. In contrast, reducing the time by 50% alone results in much weaker staining and a significant loss of signal.

To check that the effect of the SAW units were even across the entire slide we performed additional experiments where the tissue was placed exactly above or exactly between to check the SAW units (corresponding to Eridan drop zones), see Figure A3: CK AE1/AE3 staining of slides using S1700 and K5007. SAW on during 15 minutes of primary antibody incubation. Slide score (1-4) is relative (±) to control slide that have been incubated 30 minutes without SAW. H153 Small multiblock placed above (1;3;5;7;9;11) SAW unit, and between (slide 2;4;6;8;10;12) SAW units. Each section scored individually

Figure A3, page 66b: Test of SAW mixing efficiency across the drop-zones. Results from one individual experiment are shown above where small multiblock sections were placed above or between the SAW units. Results are shown for each individual section and positioned according to the position on the slide. Although there may be a trend towards a weaker staining between the drop zones (Figure A3) it is not considered significant and it is concluded that the SAW effects are even across the slides when using 3 SAW chips per slide.

The effects of SAW micro agitation were also tested on two other antibodies, the T- cell marker CD3 (A0452, 1 :100) and the proliferation marker Ki67 (MIB-1 , 1 :400). For the CD3 antibody, SAW micro agitation during a 10 minutes incubation with the polyclonal CD3 antibody gave stainings of comparable intensity (positions 1-10) with slides incubated for 20 minutes without SAW. Positions 11 and 12 with the highest energy (27dBm) scored +0.5. The reference incubated for only 15 minutes without SAW scored -1 when compared with controls.

Similar results were seen for Ki67 where 10 minutes of SAW increased the staining intensity (positions 1-12) comparable to controls incubated for 20 min. The reference incubated for only 15 minutes without SAW scored -0.5 when compared with controls.

Dilution of the primary antibody

Aim

To estimate whether SAW micro agitation is able to compensate a dilution of the primary antibody.

Method

Primary antibody incubation time will be 30 minutes with SAW units turned on. Separately stained tissues with no SAW agitation will serve as controls.

1 ) Ki67 (MIB-1 , 1 :400) on large multiblock 2) CD3 (1:100) on large multiblock

Dilution series: Exp 1 Slide 1: 1/2 dilution Slide 2: 1/4 dilution Slide 3: 1/8 dilution

Slide 4: 1/2 dilution Slide 5: 1/4 dilution Slide 6: 1/8 dilution

Slide 7: 1/2 dilution Slide 8: 1/2 dilution Slide 9: 1/4 dilution

Slide 10: 1/4 dilution Slide 11: 1/2 dilution Slide 12: 1/4 dilution Figure A4, page 66c: Dilution of the primary antibody. Results shown for Ki67 and CD3. The different SAW configurations (slide 1-12) are indicated by colour and the Power (dBm) used on the chips is also shown. Reference slides incubated with antibodies diluted either 1 :2 or 1 :4 without SAW are shown as well as two control slides incubated with antibody in the normal dilution without SAW. For the reference slides the actual IHC score is shown and for the rest of the slides the difference between that slide and the rest of the slides is given.

Results

From Figure A4 it is clear that SAW can compensate for diluting the primary antibody. Moreover, it is evident that the 80MHz 50OmW chip is the more efficient chip setup.

Find optimal SAW pulse / pause ratio Aim

To estimate whether equal staining intensity can be achieved in slides where the primary antibody incubation time has been reduced by 50% and the SAW chips are only active a fraction of the time.

Method CK AE1/AE3 staining of large multiblock slides using S1700 and K5007. The software from Advalytix allow four different settings to be tested: I) continuous, where the SAW units are constantly active II) 7/3 where the SAW units are on for 7 seconds and off for 3 seconds III) 5/5 with SAW units active for 50% of the time and IV) 3/7 with SAW units active for 3 of 10 seconds. In addition, it is possible to manually switch the SAW chips on and off if other pulse / pause are tested.

Primary antibody incubation time will be 15 minutes with SAW units turned on. Separately stained tissues with no SAW agitation incubated for 30 minutes will serve as controls. The CK antibody was used in these tests. The following pulse (time that the SAW is on) / pause (time that the SAW is off) ratios were tested and the percent of the total time with SAW mixing indicated in percent. Reference slides were compared to slides incubated 30 min. without SAW. All reference slides incubated 30 minutes without SAW were assigned a score of 2 after visual inspection. 100% Pulse / Pause ratio set to continuous (H 154)

70% Pulse / Pause ratio set to 7sec / 3sec (H 155)

50% a Pulse / Pause ratio set to 5sec / 5sec (H 156)

50% b Pulse / Pause ratio set to 30sec / 30sec (H 160) 30% Pulse / Pause ratio set to 3sec / 7sec (H 157)

25% Pulse / Pause ratio set to 1 min / 3min (H 158)

16,6% Pulse / Pause ratio set to 30sec / 2min 30 sec (H 159)

12.5% Pulse / Pause ratio set to 15sec / 1 min 45 sec (H 173)

10% Pulse / Pause ratio set to 12sec / 1 min 48 sec (H 174)

Results

The results of the pulse / pause testing are shown in Figure A6. Figure A6 is a graphic representation of the same results.

Figure A5, page 66d : Pulse / pause testing. CK antibody IHC score difference relative to a reference slide.

Figure A6, page 66e: Graphic representation of the results from the pulse pause testing.

SAW temperature effects

Some of the energy pumped into the system by SAW will eventually become heat and therefore the temperature was measured on the slides after turning on the SAW mixing. The temperature on the 80MHz 500mW chip was found to be 3°C higher than on neighbouring slides. See Figure A7, page 66f. Figure A7: Temperature measurements on SAW chips. Left) Temperature profile 80MHz, 500mW with 300ml wash buffer on the slide and temperature acquisition every 0.5 seconds with SAW being just turned of (blue) and a reference slide with no SAW (purple). Right) IHC score results from an experiment with incubation of the primary antibody under different temperatures comparing with SAW. Note that a rise in temperature during incubation with the primary antibody increases the IHC score. The increase in IHC score observed with SAW cannot be accounted for by increases in temperature but must be an effect of mixing. The details from the temperature measurements are shown below:

Test temperature Reference Difference Average 80MHz 500mW

Maximum water on slide 26,3 23,9 2,4

26.3 23,8 2,4

24,9 22,0 2,8 2,5

300Dl on slide 25,2 21 ,9 3,2

24,9 21,8 3,1 3,2

250mW

Maximum water on slide 24,5 22,2 2,3

24.1 22,0 2,1 2,2 125MHz 400mW

Maximum water on slide 25,0 21 ,7 3,3

25.4 22,0 3,4 3,4 125mW

Maximum water on slide 23,2 21,7 1,5

23.2 21,5 1 ,7 1 ,6

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Claims

CLAIMSWhat is claimed is:

1. An automated system for processing biological samples comprising: a plurality of positions, each position arranged for receiving a sample carrier (10) bearing at least one biologic sample (12); and at least one vibrator element (16) arranged to vibrate a sample carrier (10) or cover, in order to transfer vibrations to a sample or reagents applied to the sample on the carrier, wherein an actuation of the vibrator element (16) is controlled by a specific protocol controlling the processing steps of the specific sample (12) on the carrier (10).

2. An automated system according to claim 1, wherein each of the plurality of positions for sample carriers comprises at least one vibrator element arranged to vibrate a sample carrier or cover.

3. An automated system according to claim 1, wherein at least 1/3 of the plurality of positions for sample carriers comprise at least one vibrator element arranged to vibrate a sample carrier or cover.

4. An automated system according to claim 1, wherein the processing of the plurality of slides is scheduled by a multiple event scheduling program scheduling the processing steps specified in a plurality of protocols for samples present in the sample processing system.

5. An automated system according to claim 1, wherein at least one vibrator element (16) is embedded in a support element (15) arranged to come into supporting contact with a sample carrier (10).

6. An automated system according to claim 5, wherein at least two vibrator elements (16) are embedded in the support element (15) arranged to come into supporting contact with a sample carrier (10).

7. An automated system according to claim 5, wherein at least three vibrator elements

(16) are embedded in the support element (15) arranged to come into supporting contact with a sample carrier (10).

8. An automated system according to any of the claims 5-7, wherein a thin layer of a liquid is arranged between the support element (15) and the sample carrier (10).

9. An automated system according to claim 1, wherein at least one vibrator element is embedded in an element arranged to cover a sample on a sample carrier.

10. An automated system according to claim 1, wherein sample carriers may be loaded on a carousel.

11. An automated system according to claim 1, wherein the plurality of positions are arranged in a number of extractable, but normally closed, drawers and wherein one or more positions in at least one drawer comprise at least one vibrator element (16) arranged to vibrate a sample carrier or cover.

12. An automated system according to claim 4, wherein the processing of samples may be continued in all closed drawers and wherein the processing may be interrupted in a specified drawer in order to open the drawer for enabling insertion of one or more new sample carriers in the open drawer.

13. An automated system according to claim 1, wherein sample carriers may be arranged in a plurality of separate positions, and wherein transport means are arranged to execute movement of a sample carrier from one position to another, and wherein at least one of the positions is provided for a specific processing step and wherein at least one of the positions comprises a vibrator element.

14. An automated system according to claim 1 , wherein the plurality of separate positions are arranged in a compact rotary symmetric unit comprising several levels of positions enclosing or neighboring a transport means that is able to elevate and insert by translational movement a sample carrier to a specified level.

15. An automated system according to claim 4, wherein the scheduling process is repeated after insertion of a new set of carriers with new samples, each having a specified protocol.

16. An automated system according to claim 1, wherein the processing comprises the application of vibrations during at least one processing step chosen from the group consisting of washing, de-paraffϊnization, re-hydration, target retrieval, sample conditioning, enzyme application, antibody application reagent application, rinsing, chemical probe application, de-hydration, drying and mounting.

17. An automated system according to claim 1, wherein the processing comprises at least one process chosen from the group consisting of in-situ hybridization, immunohistochemistry , fluorescent in-situ hybridization and special staining.

18. An automated system according to claim 1, wherein the control of the at least one vibrator element includes controlling a start and stop of the application of vibrations.

19. An automated system according to claim 1, wherein the control of the at least one vibrator element includes controlling a frequency of the vibrations.

20. An automated system according to any of the claims 1-19, wherein the at least one vibrator element is an, SAW chip device, designed for a frequency between 50 and 150 MHz, preferably between 70 and 100MHz, such as 80 MHz,

21. An automated system according to any of the claims 1-20, wherein the at least one vibrator element is an, SAW chip device, designed for a power between 100 and 700 mW, such as between 300 and 600 mW, such as 500mW.

22. An automated system according to any of the claims 1-21, wherein the control of the at least one vibrator element includes controlling a duty cycle, a pulse length or duration of an applied vibration.

23. An automated system according to claim 22, wherein the duty cycle, or pulse length or duration of an applied vibration amount to about from 5 to 50 %, such as from 10 to 25 %, such as from 12 to 17 %, e.g. 12.5% or 16.6 % of the duration of a specific processing step applying a vibration.

24. An automated system according to any of the claims 1-23, wherein the control of the at least one vibrator element includes sweeping a frequency of the vibrations.

25. An automated system according to any of the claims 1-23, wherein the control of the at least one vibrator element includes modulating a frequency of the vibrations.

26. An automated system according to any of the claims 1-23, wherein the control of the at least one vibrator element includes controlling an amplitude of the vibrations.

27. An automated system according to claim 4, wherein a scheduling and control of a vibrator element is related to the protocol defining the processing of the sample to be vibrated.

28. An automated system according to any of the claims 1-27, further comprising a vibration controller that regulates electrical power to the at least one vibrator, the vibration controller being controlled in dependence of the specific protocol.

29. A method of automated processing of biological samples comprising the steps of: establishing an automated sample processing system having an automated process operation capability that causes automated process operation events through robotic sample process functions; loading a plurality carriers with biologic samples in the automated sample processing system; loading or accessing data enabling the sample processing system to define at least one protocol for the control of the sample processing of each of the loaded samples; and executing the sample processing using at least one vibrator element to enhance the processing under at least one processing step.

30. A method according to claim 29, wherein the at least one processing step comprises a wash step for which the washing effect is enhanced by vibrations from the vibrator element.

3 1. A method according to claim 29, wherein the at least one processing step comprises a de-paraffmization step for which the de-paraffmization is enhanced by vibrations from the vibrator element.

32. A method according to claim 29, wherein the at least one processing step comprises a mixing step, for which the mixing is enhanced by vibrations from the vibrator element.

33. A method according to claim 29, wherein the at least one processing step comprises a mounting step that includes an application of a viscous polymer, the viscosity of the polymer being decreased so that the polymer may easily flow over a desired area under the effect of vibrations from the vibrator element.

34. A method according to claim 29, wherein the at least one processing step comprises a heating step for which a homogenization of sample temperature is enhanced by vibrations from the vibrator element.

35. A method according to claim 29, wherein the at least one processing step comprises a re-hydration step for which the re-hydration is enhanced by vibrations from the vibrator element.

36. A method according to claim 29, wherein the at least one processing step comprises a target retrieval step for which the target retrieval is enhanced by vibrations from the vibrator element.

37. A method according to claim 29, wherein the at least one processing step comprises application of an enzyme or an antibody for which the application of the enzyme or antibody is enhanced by vibrations from the vibrator element.

38. A method according to claim 29 wherein the vibrator emits acoustic waves of a wavelength adapted to the at least one processing step that is assisted by the waves.

39. A method according to claim 29 wherein the vibrator emits acoustic waves of a wavelength that is smaller than or the same size as the sample carrier.

40. A method according to claim 29 wherein the sample processing comprises a specific processing step wherein vibrations are applied to the sample during at least a fraction of the time during the specific processing step and wherein the processing time is reduced at least 25 %, such as to about 50 % of the normal or standard processing time for the specific step.

41. A method according to claim 29 wherein the sample processing comprises an incubation step wherein vibrations are applied to the sample during at least a fraction of the time during the incubation step and wherein the incubation time is reduced by at least 25%, such as to about 50 % of the normal or standard incubation time.

42. A method according to claim 41 wherein the vibrations are applied at least 12 %, such as at least 16.6%, such as at least 25%, such as at least 30%, such as at least 50 % of the time during the incubation step.

43. A method according to claim 29 wherein the reagents used for the sample processing has been diluted by a factor of about 2 to 4, and wherein application of vibrations compensate for the diluted reagents, so that the desired chemical reaction is obtained.