Abstract:
An apparatus and method for applying reagents to tissue mounted on slides, in the field of histology is disclosed. The apparatus holds a number of slides, in slide trays, which are loaded onto the apparatus. Each slide tray forms a batch of slides, with all the batched forming a group. The apparatus holds a number of reagents, grouped into a first group comprising bulk type reagents, and a second group comprising antibodies or probes, and detection systems, for identifying elements of the tissue. A group fluid dispenser, in the form of a robot arm, dispense reagents to the group of slides. Each batch of slides has its own batch fluid dispenser to dispense reagents onto the batch of slides. In on embodiment, the group fluid dispenser dispense antibodies, probes, detection reagents to all slides depending on the protocol defined for each slide, and the batch dispensers dispense bulk reagent to each batch, thus freeing the group fluid dispense from dispensing bulk reagents to all slides.

Description:
This application is a National Stage Application of PCT/AU2009/000771, filed 16 Jun. 2009, which claims benefit of Serial No. 2008903057, filed 16 Jun. 2008 in Australia and U.S. Ser. No. 61/061,767, filed 16 Jun. 2008 and which applications are incorporated herein by reference. To the extent appropriate, a claim of priority is made to each of the above disclosed applications. 
     FIELD 
     The present invention relates to improved apparatus and methods for staining tissue mounted on microscope slides. 
     BACKGROUND 
     A number of techniques have been developed that involve placing a biological sample onto a substrate. Examples of these techniques include placing tissue samples from biopsies onto microscope slides, and micro-array analysis of samples. In histology, biological samples are attached to a microscope slide and stained to enhance the visibility of features of the tissue. Examples include routine staining, using haematoxylin and eosin stains to improve visibility of cell walls and the cell nucleus. In advanced staining, antibodies are applied to the tissue and then stained to identify the presence or absence of particular proteins, which may be indicative of disease. Other reagents may be applied to tissue such as RNA/DNA probes, which, during a sequence of reactions on the tissue, may bind to DNA in the cell nucleus. The hybridised DNA may then be stained to identify presence or absence of DNA of interest. 
     The number of proteins that may be used in investigation, research or diagnosis is large, and ever increasing. Similarly, the number of genes of interest is also increasing. If an automated instrument is to apply the wide range of reagents used in diagnosis or research, then the instrument must have flexibility. However, it is also important that samples are completed quickly and efficiently. 
     One instrument that is used to test slides is the Bond-max automated advanced staining instrument sold by Leica Microsystems, and described in application number WO 04/001390A1: BIOLOGICAL REACTION APPARATUS WITH DRAINING MECHANISM. A robotic arm is used to dispense reagent onto the slides located onto the instrument. The slides are loaded into the instrument in batches, which provides flexibility, as the instrument can start processing a first batch before a second batch is loaded. The capacity of the instrument, and its ability to process multiple batches simultaneously, is limited by the number of batches, the protocols and processing time of each slide, and the speed of the robotic arm in completing its tasks. There is a need to increase the flexibility of automated staining instruments, such as by decreasing processing time for slides, including batches of slides. 
     SUMMARY 
     In one aspect, there is provided an instrument for applying reagents to a group of microscope slides, having: a plurality of slide supports; a first set of one or more reagent containers; a second set of one or more reagent containers; a group fluid dispenser for dispensing fluid drawn from one or more reagent containers of the first set of reagent containers, and a batch fluid dispenser for dispensing fluid drawn from one or more reagent containers of the second set of reagent containers. 
     In one form the slides are grouped into a plurality of batches. 
     In one form the group fluid dispenser is mounted on a robotic arm to dispense reagent to all the slides. 
     In one form the batch fluid dispenser is mounted to a robotic arm to dispense fluid onto one of the batches of slides. 
     In another aspect, there is provided an apparatus for dispensing fluid onto a plurality of substrates, comprising: a group of substrates, formed from one or more sub-groups of substrates; a group dispenser adapted to dispense fluid onto the entire group of substrates; and one or more additional sub-group fluid dispensers adapted to dispense fluid onto one or more sub-groups of substrates. 
     Preferably, each of the one or more sub-group fluid dispensers is located adjacent to the one or more sub-groups of substrates upon which it is to dispense fluid. 
     In another aspect, there is provided a method of dispensing reagent onto a group of slides comprising the steps of applying reagent from a group fluid dispenser to slides in the group of slides; and applying reagent from a plurality of batch fluid dispensers to a corresponding plurality of batches of slides, where the group of slides consists of two or more batches of slides. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a schematic drawing of an apparatus having multiple fluid dispensers; 
         FIG. 2  is a schematic drawing of a portion of the apparatus of  FIG. 1  showing a slide staining assembly and a fluid dispenser; 
         FIG. 3  is a schematic drawing of a fluid dispenser and slide staining assembly of the apparatus of  FIG. 1 ; 
         FIG. 4  is a schematic drawing of a cross section of a fluid dispenser head of the fluid dispenser shown in  FIGS. 2 and 3 . 
     
    
    
     DETAILED DESCRIPTION 
     The apparatus  10  shares many common elements and operational methods with the Bond-max instrument sold by the applicant, and in part described in international patent application No. WO 04/001390A1, titled BIOLOGICAL REACTION APPARATUS WITH DRAINING MECHANISM, which is hereby incorporated by reference. Also incorporated by reference is the Bond-max user manual, which also describes the operation of the Bond-max instrument. If there is a conflict between the aforementioned documents, the user manual should be read as the correct description. 
     The apparatus  10  shown in  FIG. 1  is a slide-staining instrument used to stain tissue mounted to microscope slides, but may also be used to apply fluids to substrates, such as micro array plates or other substrates used for biological testing. The apparatus  10  includes a group fluid dispenser  11  mounted to a robot arm  14 , a first set of reagents, in containers  15 , a second set of reagents  16 , and slide staining assemblies  17 ,  18  and  19 . Also shown are syringe pumps  21 , in fluid connection with reagent containers  15 . 
     The apparatus  10  is operated by a computer (not shown) used to instruct the apparatus of the protocols to be applied to each slide. A computer internal to the apparatus  10  receives information on the protocols to be applied to the slides, and controls the various functions of the apparatus  10 , including operation of the robotic arm  14 , slide staining assembly  17 ,  18  and  19 , and reagent dispensation. In relation to systems not connected to the operation or function of the batch and group fluid dispensers, such as slide loading, slide identification, protocol identification, slide heating, and covertile opening and closing, the apparatus  10  operates in the same way, and uses the same or similar components, to a Bond-max instrument as sold by the applicant. Where a feature of a fully functioning instrument is not described in the present application, it can be taken that the feature would operate as per a standard Bond-max instrument sold by the applicant and described in the documents incorporated by reference herein. 
     Each slide staining assembly  17 ,  18  and  19  has a corresponding batch fluid dispenser  30 , as shown in  FIGS. 2 and 3 . 
     In  FIGS. 2 and 3 , the slide staining assembly  17  (which in this embodiment is identical to the other slide staining assemblies  18  and  19 ) is shown, having ten slide supports  32  under a cover  33 . In operation a batch of slides is loaded into one of the slide staining assemblies  17 ,  18  and  19  on a Bond slide tray  41 . The slides in the tray  41  are located to correspond with the location of the slide supports  32 , and therefore in operation, a slide may be located on each slide support  32 . Each slide support  32  includes a heater element, for heating the slide and tissue, and has a locating mechanism for moving a covertile on each slide in the same way as Bond-max instruments operating at the time of filing this application. 
     The slide staining assembly  17  is located adjacent to a batch fluid dispenser  30  as shown in  FIGS. 2 and 3 . The batch fluid dispenser  30  includes a rail  34  along the side of the slide staining assembly  17 , and a dispense head  36  ( FIG. 4 ) adapted to move along the rail  34  so that fluid is able to be dispensed on each of the slides in the assembly  17 . An energy chain device is used to drive the dispense head  36  along the length of the rail  34  to the slide required. In one example, the dispense head  36  is moved along the rail  34  from one end to the other, stopping at a position adjacent each slide, and dispensing reagent onto the slide to interact with the tissue mounted thereon. The energy chain houses a flexible fluid conduit, which terminates in the dispense head  36 , to direct fluid onto the slide. The conduit is attached to a syringe pump  21  dedicated to the specific dispenser. Thus in the instrument shown, each of the three batch fluid dispensers  30  has its own syringe pump  21 . Fluid may be drawn from a number of reagent containers  15 . In the present example each of the aforementioned reagent containers  15  has its own manifold. Each manifold is connected to all the syringe pumps  21  via individual conduits. Each syringe pump  21  has a selectable valve structure that allows the syringe pump  21  to fluidly connect to the desired manifold, and thus access the desired fluid type. For example, if dispenser  30  was required to dispense alcohol, the valve would move to open the connection to the conduit from the alcohol reagent container, and the syringe pump  21  would draw alcohol through the conduit into the syringe until the desired volume was attained. The syringe pump  21  then closes the valve to the alcohol container, and opens the connection to the batch fluid dispenser  30 . The syringe pump  21  then pushes the desired volume of fluid along the conduit to the batch fluid dispenser  30 , where alcohol is dispensed onto the appropriate slide  32 . 
     The amount of fluid drawn into the syringe pump  21  is determined by the volume of fluid in the conduit, as well as the number of slides requiring fluid and the volume of dispense per slide. The batch fluid dispenser  30  can therefore dispense a reagent onto one or more slides, by stopping adjacent each slide location. Once the fluid has been dispensed onto the slides, the syringe pump  21  will be empty, but fluid will be retained in the conduit. In order to flush the conduit fluid, the batch fluid dispenser  30  moves to the end of the slide staining assembly  17 ,  18 ,  19 , where a washing station (not shown) is located. The washing station has an aperture to receive fluid from the batch fluid dispenser  30 , whereupon the fluid received by the washing station is directed to a waste container. Typically, in the present embodiment, the syringe pump  21  will push a volume of the next fluid to be dispensed through the conduit and to waste to wash out the previous fluid. Once old fluid has been flushed, the batch fluid dispenser  30  may move to the slides again and dispense reagent as required. 
     In certain situations, some of the fluids are incompatible, for example, they may not be miscible. In such a situation where it is determined that the new fluid may not satisfactorily wash out the old fluid from the conduit, an intermediary fluid may be used. 
     An example of incompatible fluids would be distilled water and Dewax fluid. However, alcohol is miscible in both and therefore would be used as an intermediary fluid to remove one fluid before application of a new fluid. 
     In operation of the apparatus  10 , having slides each with a tissue sample mounted thereon, each slide will have a protocol assigned to it, based on the type of reagent (antibody or probe) to be applied to the tissue sample on the slide. A typical protocol has an initial fluid dispense for each slide, application of further fluid, for example a Dewax fluid for removing wax from a sample, application of Epitope retrieval fluid, application of specific reagent for that sample, various detection fluids (stains), all or some of which may be interspersed with the application of wash fluid to remove the previous fluid. 
     Application of the fluids may in some cases need to be done at specific intervals, for example antibodies may need to be left on a sample for a specific amount of time, before being washed off. Staining of tissue can be affected if fluid is left on for too long, or if fluid levels are not replenished causing tissue to dry out. There are also times when no fluid should be dispensed onto a slide, for example incubation time, and therefore the robotic arm and dispenser may remain idle for a period of time before the next operation is required. Thus timing of the dispensation of fluid can be critical, which can cause scheduling issues when running two or more batches simultaneously. 
     It is not necessary to load all three trays at once. It is possible to load a first tray having between one and ten slides, into a staining assembly  17 ,  18 ,  19 , and start the operation of the instrument. At any time, other trays may be loaded, which is defined as batch processing. That is, the apparatus  10  deals with each tray of slides as batches, and can begin operations on a newly entered batch before completing operations on a previously loaded batch. In the Bond-max instrument as in the apparatus  10 , there are three slide staining assemblies  17 ,  18  and  19  that represent three batches when loaded, however in the Bond-max instrument there is only one dispenser capable of dispensing fluid onto all slides in the slide staining assemblies. Thus in the Bond-max instrument, one robot must undertake all fluid dispensing tasks on all batches of slides. In apparatus  10 , as described below, the operation of the group fluid dispenser  11  has been reduced by the use of specific batch fluid dispensers  30  plumbed into reagent containers  15 . 
     In operation, the fluid dispenser  11  on robotic arm  14  moves along the line of slides in a batch within a slide staining assembly  17 ;  18 ,  19  and undertakes the required dispensation of fluid on all slides in the batch before moving to another slide assembly  17 ,  18 ,  19 . 
     In the apparatus  10 , reagent containers  16  may contain reagents such as detection reagents (conjugating reagents and stains), antibodies and probes. Given their small volumes and high value, these containers are not plumbed into any dispenser, as the fluid lines and washing would waste too much reagent. Instead, the group fluid dispenser  11  has a pipette used to withdraw reagent directly out of the required reagent container  16 . In the apparatus  10  shown, over thirty different reagent containers  16  may be loaded into the apparatus  10 , and these typically include one detection system consisting of nine reagents, with the remainder being various antibodies or probes. It is therefore possible to apply a different antibody to most slides. In operation, it is necessary to rinse out the group fluid dispenser  11  between dispensations of different antibodies, to prevent cross contamination. Therefore the robot arm  14  may need to move from one of the reagent containers  16  to draw fluid, then to a slide to dispense fluid, then to a wash station to wash the dispenser pipette, then to another reagent container  16 , then to a new slide. Thus the robot arm may be in use for a considerable period of time to dispense fluid onto ten slides in a single batch. During this period, dispensation of other fluids onto other batches cannot take place from the robot arm  14  and dispenser  11 , as the group fluid dispenser is fully utilised. In the apparatus  10 , batch fluid dispensers  30  as shown in  FIG. 2  are used to dispense reagents from reagent containers  15  onto the slides. For example, while group fluid dispenser  11  is dispensing antibodies onto the slides in slide staining assembly  17 , one of the batch fluid dispensers  30  may dispense reagent onto the slides in another batch, or slide staining assembly  18  and/or  19 . 
     Thus in operation, the group fluid dispenser  11  can dispense reagent onto each slide in a batch, while the dispensers  30  associated with each slide staining assembly  17 ,  18  and  19  can move from side to side in their own batch and dispense reagent. This may be done simultaneously as required, thus freeing the group fluid dispenser  11  and robot arm, and allowing the batches to be completed more quickly. The group fluid dispenser  11  now has fewer dispenses to make, providing greater free time between dispenses. This makes scheduling a new batch much easier as the gap between times the group fluid dispenser (dispenser  11 ) is in use is larger, and the time required by the group fluid dispenser  11  to dispense some reagents may be shorter, as some dispensing may be undertaken by the batch dispensers (dispensers  30 ) to undertake a dispense on all sides is shorter. Thus, it is much easier to interleave the operations of the group fluid dispenser  11  in one batch into the operations of the group fluid dispenser  11  in another batch, and subsequently this reduces the time to complete three batches. 
     Typical reagents that the batch fluid dispenser  30  would dispense include reagents such as deionised water, alcohol, buffer fluid, Epitope retrieval fluid, and Dewax fluid. These fluids may be called bulk fluids, as they typically are stored in reagent containers  15  of between 2000-5000 ml. In contrast, the reagent containers  16  typically hold between 5-30 ml. 
     In the present embodiment, the batch fluid dispensers  30  use an optical sensor to detect when they are in the correct position. The batch fluid dispenser  30  is moved by a stepper motor, which is controlled to step a predetermined number of steps from one dispense position to the next. Once the motor has driven the batch fluid dispenser  30  the assigned number of steps, the system checks the optical sensor to determine whether the batch fluid dispenser  30  is in the correct position relative to the slide. If the batch fluid dispenser  30  is not in the correct position, the system moves the batch fluid dispenser  30  forward along the track  34  until it detects that it is in the correct position. While moving along track  34 , the batch fluid dispenser  30  is supported in the track  34  by guide wheels  42 . 
     The apparatus  10  may operate with one, two or three slide trays loaded into the three staining assemblies. Other examples of apparatus may not be limited to three slide staining assemblies, and for example may have two, or more depending on the throughput and size of apparatus required.