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WO2010077975A1 - Appareil d'isolement du dessous d'une lame - Google Patents

Appareil d'isolement du dessous d'une lame Download PDF

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Publication number
WO2010077975A1
WO2010077975A1 PCT/US2009/068303 US2009068303W WO2010077975A1 WO 2010077975 A1 WO2010077975 A1 WO 2010077975A1 US 2009068303 W US2009068303 W US 2009068303W WO 2010077975 A1 WO2010077975 A1 WO 2010077975A1
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WO
WIPO (PCT)
Prior art keywords
slide
plate
housing
microscope slide
pressure reducer
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2009/068303
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English (en)
Inventor
Zachary Daniel Wojtowicz
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Ventana Medical Systems Inc
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Ventana Medical Systems Inc
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Filing date
Publication date
Application filed by Ventana Medical Systems Inc filed Critical Ventana Medical Systems Inc
Publication of WO2010077975A1 publication Critical patent/WO2010077975A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/34—Microscope slides, e.g. mounting specimens on microscope slides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/36—Embedding or analogous mounting of samples

Definitions

  • the present disclosure concerns an automated microscope slide processing apparatus and method for its use.
  • Molecular pathology is the molecular level examination of DNA, mRNA and proteins that cause or otherwise are associated with disease. This examination can elucidate important information about patient diagnosis, prognosis, and treatment options. Molecular pathology generally is divided into two main areas: (i) analysis of DNA, mRNA, and proteins in intact cells (in-situ), and (ii) analysis of these biological materials after they have been extracted from tissues. The first category allows the pathologist or scientist to study the histopathologic architecture or morphology of the tissue specimen microscopically while the nucleic acid or proteins are assayed.
  • ISH immunohistochemistry
  • HC histochemistry
  • EHC enzyme histochemistry
  • IHC utilizes antibodies that bind specifically with unique epitopes present only in certain types of diseased cellular tissue. IHC highlights morphological indicators of disease states by selective staining; this requires a series of treatment steps conducted on a tissue section or cells (e.g. blood or bone marrow) mounted on a glass. Typical steps include pretreating the tissue section to remove paraffin and to reduce non-specific binding; retrieval of antigens masked by cross-linking of proteins by chemical fixatives; antibody treatment and incubation; enzyme-labeled, secondary antibody treatment and incubation; substrate reaction with the enzyme to produce a fluorophore or chromophore, thereby highlighting areas of the tissue section having epitopes binding with the antibody; counterstaining; and the like.
  • tissue section or cells e.g. blood or bone marrow
  • ISH analysis which relies upon the specific binding affinity of probes with unique or repetitive nucleotide sequences, requires a similar series of process steps with many different reagents. Varying temperature requirements further complicate ISH analysis.
  • U.S. Patent No. 5,654,200 to Copeland et al. describes an automated biological processing system comprising a reagent carousel cooperating with a sample support carousel to apply a sequence of preselected reagents to each of the samples with interposed mixing, incubating, and rinsing steps.
  • This patented automated biological processing system which is available from Ventana Medical Systems, Inc. of Arlington, Arizona, includes a slide support carousel having a plurality of slide supports.
  • a drive mechanism engages the slide support carousel for consecutively positioning each of a plurality of slide supports in a reagent receiving zone.
  • the reagent carousel comprises a plurality of reagent container supports.
  • the drive mechanism engages the reagent carousel to rotate the carousel to position a preselected reagent container support and associated reagent container in a reagent supply zone.
  • the apparatus has a reagent delivery actuator mechanism configured to engage a reagent container positioned on a container support in the reagent supply zone and to initiate reagent delivery from the reagent container to a slide that is on a slide support in the reagent receiving zone.
  • the slide support comprises a metal plate mounted on a molded plastic base.
  • An electrical resistance heater is mounted in direct contact to the underside of the metal plate. Corner pins locate a specimen carrying glass slide on the surface of the metal plate.
  • the metal plate has a top surface that is essentially flat and smooth. Flatness and smoothness facilitate glass plate position stability and thermal conduction uniformity. This slide support allows for occasional processing failures, and further refinement is needed to alleviate these processing failures.
  • the disclosure concerns an automated slide processing device, and more particularly to an improved slide support.
  • a microscope slide containing a sample usually a tissue sample, on one side is placed on a platform, sample side up.
  • water and other fluids used to process the sample may contaminate the backside of the slide, inhibiting analysis of the processed slide and necessitating post processing backside cleaning. Additionally, such fluids may contact the hot metal plate and boil, causing localized high pressure between the slide support and the slide, thereby dislocating the slide. Slide dislocation may compromise further processing of that one slide, or worse yet, result in a domino effect where a first dislocated slide contacts a neighboring slide, thereby causing that slide to dislocate, and so forth.
  • Disclosed embodiments of the present apparatus and method for its use address these deficiencies by sealing the area of the slide that is over the plate, thereby preventing processing fluids from entering the area between the slide and the plate.
  • This provides several advantages, including: mitigating the need to clean the backside of the slides after processing; preventing slide dislocation from unwanted vaporization; and improving the contact between the slide and the plate, thereby affording more efficient conductive heat transfer.
  • the apparatus is equipped with a platform, shaped and sized to receive the microscope slide, and a seal circumscribing the platform upon which the microscope slide rests.
  • a pressure reducer is actuated to at least partially, if not substantially completely, evacuate the volume defined by the space between the platform and the bottom surface of the microscope slide.
  • the resultant pressure differential between the ambient pressure conditions above the microscope slide and the space between the microscope slide and the platform create a net force urging the microscope slide toward the platform, compressing the seal and forcing the sample-free area into tight contact with the platform.
  • the pressure reducer may engage only when a microscope slide is placed on the platform.
  • the apparatus may include a sensor for sensing the microscope slide and a mechanism for fluidly connecting the pressure reducer with the volume defined by the slide bottom surface, the seal, and the plate, thereby sealing the slide bottom surface.
  • the sensor may signal actuation of the pressure reducer.
  • the sensor may send an electronic signal to a controller or a relay that, in turn, opens a valve or otherwise engages the pressure reducer, thereby fluidly connecting the pressure reducer with the volume between the plate and slide.
  • a sensor may be a normally-closed, mechanically actuatable valve, such as a poppet valve, actuated by placing the slide on the platform.
  • the seal is formed from a pliable, waterproof, heat- resistant, and chemical resistant material.
  • Exemplary seal materials include Viton®, Kalrez®, Chemraz®, Viton® ExtremeTM ETP, Simriz®, nitrile (buna-n), neoprene, silicone, polyurethane, and Teflon encapsulated elastomers.
  • the physical design of the seal may be a custom-engineered, continuous ridge sized to circumscribe the plate and extend vertically above the top of the plate.
  • the seal may be an off-the-shelf design, such as an O-ring.
  • the platform may incorporate a thermally conductive plate operatively associated with a temperature control unit for controlling the temperature of the slide and sample during processing.
  • the temperature control unit may comprise a heater and temperature sensor.
  • the plate may rest on a housing formed from a thermally insulating material.
  • the thermally insulating material may be thermoset plastic or vulcanized rubber.
  • the housing may also define a cavity to contain any electrical components or other connections necessary for device operation, such as for operation of the temperature control unit.
  • the housing may contain a groove, circumscribing the cavity, for holding a seal, such as an O-ring.
  • the housing also may include a fastening mechanism to fasten the housing to the automated processing unit. For instance, it may contain a clamping mechanism or plural apertures for receiving fasteners.
  • the plate may be sealably associated with the housing.
  • the seal between the housing and the plate may be accomplished by coupling a plate border to the plate edges, thereby inhibiting processing fluids from flowing into the cavity.
  • the plate border may comprise a pliable, substantially waterproof material with features designed to seal against the housing. Because the plate may reach a temperature of up to 300 0 C, it may be desirable for the plate border to be heat resistant up to at least 300 0 C as well, more typically heat resistant up to about 200 0 C, and even more typically heat resistant up to a temperature of about 100 0 C or less.
  • the housing has an indentation extending continuously around the upper outer wall of the cavity defined by the housing.
  • the plate border has a lip extending downward with a ridge on the inner surface for sealing against the indentation in the housing.
  • the housing may include physical restraints for preventing lateral movement of a microscope slide relative to the plate.
  • such restraints may be plural posts extending upwardly and substantially orthogonally from the bottom surface of the slide support.
  • the posts may be desirable for the posts to have a height less than the cross-sectional thickness of the microscope slide.
  • the pressure reducer may be operatively connected to a manifold.
  • the manifold may be a separate piece, incorporated in the housing, or incorporated in the plate. The manifold is contained within the area defined by the seal and provides a path for fluidly connecting the pressure reducer and the volume to be evacuated.
  • the pressure reducer may be fluidly connected to the cavity and the cavity may be fluidly connected to the volume to be evacuated via one or more apertures in the housing or the plate.
  • the aperture(s) may contain a valve(s) for controlling the fluid connectivity between the cavity and the volume to be evacuated, or a valve may control the pressure reducer itself.
  • FIG. 1 is a perspective view of one embodiment of a disclosed slide support with a microscope slide in place.
  • FIG. 2 is a plan view of one embodiment of a disclosed slide support without the microscope slide.
  • FIG. 3 A is a cross-sectional view of one embodiment of a disclosed slide support with a microscope slide in place, showing the volume to be evacuated between the slide and the plate.
  • FIG. 3B is a cross-sectional view of one embodiment of a disclosed slide support with the volume evacuated.
  • FIG. 4 is a cross-sectional view of one disclosed embodiment of a plate.
  • FIG 5 is a plan view of one embodiment of a plate and manifold.
  • FIG. 6 is a perspective view of one embodiment of the apparatus having a poppet valve.
  • FIG. 7 is a side view of one embodiment of the apparatus having a clamping mechanism.
  • FIG. 8 is a perspective assembly view of the automated processing unit.
  • “Lateral movement of the microscope slide” refers to movement in the direction of the microscope slide edges.
  • Pressure reducer refers to any device, utility, or resource capable of reducing the pressure of a substantially bounded fixed volume. Exemplary pressure reducers include vacuum pumps and flowing liquid aspirators.
  • Poppet valve refers to a normally closed valve that rises perpendicularly to its seat.
  • Sensor refers to a component or feature that responds to a physical stimulus with a mechanical action and/or electric, electronic, or pneumatic signal. Physical stimulus, as used in this definition, includes mechanical stimulus, optical stimulus, capacitive stimulus, or other stimulus effective to achieve disclosed functions.
  • Stain refers to any biological or chemical entity that, when applied to targeted molecules in tissue, renders the molecules detectable under a microscope.
  • Substantial portion of a surface refers to a continuous area of a surface containing about 50% or more of the total area of the surface.
  • tissue refers to any collection of cells that can be mounted on a standard glass microscope slide including, without limitation, sections of organs, tumors sections, bodily fluids, smears, frozen sections, cytology preps, and cell lines.
  • Treating refers to application of a stain or other detectable label to a tissue as well as other processes associated with such application including, without limitation, heating, cooling, washing, rinsing, drying, evaporation inhibition, deparaffinization, cell conditioning, mixing, incubating, and evaporation.
  • FIG. 1 shows a perspective view of one embodiment of the presently disclosed apparatus 10.
  • FIG. 1 shows microscope slide 12 resting on the apparatus 10.
  • Apparatus 10 comprises a slide support 14 and a pressure reducer 16 operatively associated with the slide support.
  • Slide support 14 has a top surface 18 for receiving the microscope slide 12.
  • Top surface 18 is circumscribed by a seal 20.
  • the slide support 14 may include physical restraints for locating and preventing lateral movement of a microscope slide relative to the plate.
  • FIG. 1 shows plural posts 26 extending upwardly and substantially orthogonally from the bottom surface 28 of the slide support 14. To prevent capillary forces from wicking reagents or other fluids from the microscope slide 12, it may be desirable for the height of the posts 26 to be less than the cross-sectional thickness of the microscope slide when the slides rest on the slide support 14.
  • FIG. 1 shows an apparatus 10 sized and shaped to accommodate a rectangular microscope slide 12. Slides are not limited to rectangular slides. The size and/or shape of the apparatus can be modified to accommodate whatever size and/or shape slide is used or needed in a given system.
  • FIG. 1 shows the apparatus 10 having fastening mechanism 30 for mounting the apparatus to the automated processing unit (not shown).
  • the fastening mechanism 30 shown in FIG. 1 comprises plural apertures 32 and fasteners 34.
  • one embodiment of apparatus 10 further comprises a sensor 36. When a microscope slide 12 is placed on the slide support 14, sensor 36 detects the microscope slide, thereby actuating the pressure reducer 16. Any appropriate sensor capable of detecting the physical presence of an object may be employed.
  • sensors include, without limitation, optical sensors, ultrasonic sensors, inductive sensors, capacitive sensors, mechanical, or mechanical/electrical sensors.
  • the pressure reducer 16 is controlled by valve 37.
  • the sensor 36 when sensing a microscope slide 12, may send an electronic signal to a controller or a relay which, in turn, opens valve 37.
  • sensor 36 may be a normally-closed, mechanically actuatable valve, actuated by the microscope slide 12 when it is placed on the slide support 14.
  • Such a sensor may be a poppet valve.
  • the slide support 14 includes a housing 38 and a plate 40.
  • the pressure reducer 16 is fluidly connected with the volume 42 defined by the bottom surface 24, the top surface 18 and the seal 20.
  • the volume 42 is evacuated to an extent sufficient to create a pressure differential, forcing the seal 20 to compress between the plate 40 and the microscope slide 12, thereby sealing a substantial portion 22 of the bottom surface 24 and forcing the bottom surface of the microscope slide into tight contact with the top surface 18 of the slide support, as shown in FIG. 3B.
  • Such mechanisms may include, without limitation, a three-way valve vented to ambient air or connected to a positive pressure source. Re-pressurization may be controlled automatically, semi- automatically, or manually.
  • the seal 20 desirably seals against smooth, hard surfaces, such as the glass surface of most microscope slides. Additionally, the slide processing environment is a wet environment that uses various chemical reagents. Tissue processing also may be facilitated by heating the microscope slide.
  • the seal 20 can be formed from a pliable, substantially waterproof, heat-resistant, and chemical-resistant material. Substances for making such seals can include, without limitation, Viton®, Kalrez®, Chemraz®, Viton® ExtremeTM ETP, Simriz®, nitrile (buna-n), neoprene, silicone, polyurethane, and Teflon encapsulated elastomers.
  • the physical design of the seal 20 may be a custom engineered continuous ridge, sized to circumscribe the top surface 14 and extend vertically above the top surface. Alternatively, seal 20 may be an off-the-shelf design, such as an O-ring.
  • the plate 40 can be thermally conductive and operatively associated with a temperature control unit.
  • a thermally conductive plate may comprise any material with sufficient thermal conductivity to afford efficient and uniform heat transfer to the microscope slide 12. Such materials include, without limitation, copper, silver, aluminum, iron, and alloys thereof, such as brass and stainless steel.
  • the plate also may have a plurality of slots or channels in the top surface of the plate as disclosed in U.S. Patent No. 7,425,306, which is incorporated by reference.
  • FIGS. 3 A and 3B show plate 40 operatively associated with temperature control unit 44.
  • Temperature control unit 44 comprises heater 46 and temperature sensor 48. Temperature control units for such purposes have been described in detail in U.S. Patent No. 6,296,809, which is incorporated herein by reference. A variety of commercially available heaters and temperature sensors may be employed for this purpose.
  • the neighboring slide supports can be thermally isolated and the electrical components can be protected.
  • the plate 40 may rest on a housing formed from thermally insulating material.
  • the temperature control unit and electrical connections can be protected from processing fluids.
  • FIG. 3A shows temperature control unit 44 and electrical connections 50 contained in a sealed volume.
  • the slide support 14 comprises a housing 38 and a plate 40.
  • the housing 38 defines a cavity 52.
  • the plate 40 is sealably associated with the housing 38.
  • a plate border 54 is coupled to the plate edges 55.
  • the plate border 54 may be or include a pliable, heat resistant, water- resistant material, such as Viton®, Kalrez®, Chemraz®, Viton® ExtremeTM ETP, Simriz®, nitrile (buna-n), neoprene, silicone, polyurethane, and Teflon encapsulated elastomers. Because the plate 40 may reach temperatures up to 100 0 C, plate border 54 also can be heat-resistant up to 100 0 C.
  • the plate border 54 has a lip 56 extending downwardly with a ridge 58 on the bottom inner surface 60 of the lip. Ridge 58 coordinates with an indentation 62 in the housing 38, thereby substantially sealing the plate to the housing.
  • the housing bottom 64 has a groove 66 circumscribing the cavity 52, for receiving a seal 68.
  • the seal 68 is compressed in the groove 66 against the automated processing unit, thereby sealing the volume defined by the housing, the plate 40, and the automated processing unit.
  • the plate 40 can be protected from the processing environment.
  • One method for protecting plate 40 is to coat the thermally conductive plate at least partially or substantially completely with a protective coating.
  • a protective coating include, without limitation, ceramic, hard anodization, epoxy, Teflon, and silicone.
  • FIG. 4 is a cross-sectional view of coated plate 72 comprising thermally conductive plate 40 and protective coating 74.
  • the pressure reducer may be operatively associated with a manifold.
  • FIG. 5 shows plate 40 with manifold 76.
  • Manifold 76 may be a separate piece, incorporated in the housing 38 or in the plate 40, and contained within the area defined by the seal 20.
  • Manifold 76 comprises orifices 78 that are fluidly connected to the pressure reducer 16.
  • Using a manifold has the advantage of improving temperature uniformity in plate 40 by minimizing localized effects on the heat conduction profile.
  • FIG. 6 illustrates an embodiment where the pressure reducer 16 is fluidly connected to the cavity 52 and the cavity is fluidly connected to the volume 42 to be evacuated via an aperture 80 in the plate.
  • a poppet valve 82 is positioned in aperture 80 for controlling the fluid connectivity between the cavity 52 and the volume 42 to be evacuated.
  • FIG. 7 is a side view showing an alternate mechanism for fastening the apparatus 10 to the automated procession unit 70.
  • FIG. 7 shows a lip 84 coordinating with the apparatus 10 and a latch 86 for holding the apparatus to automated processing unit 70.
  • other mounting mechanisms known, in the art and capable of exerting adequate force to compress the seal 68 may be employed.
  • Apparatus Disclosed slide support embodiments can be components of an automated processing device.
  • Embodiments of automated staining devices are disclosed in various patents assigned to Ventana Medical Systems, Inc., including 5,595,707, 5,654,200, 6,296,809, 6,582,962, 7,270,785, 7,303,725, 7,396,508, each of which is incorporated herein by reference. A brief, general description of suitable automated processing devices follows.
  • the automated processing unit 70 is designed to automatically stain or otherwise treat tissue mounted on microscope slides with nucleic acid probes, antibodies, and/or reagents associated therewith in the desired sequence, time, and temperature. Tissue sections so stained or treated are then to be viewed under a microscope by a medical practitioner who reads the slide for purposes of patient diagnosis, prognosis, or treatment selection.
  • the automated processing unit functions as a module of a system further comprising a host computer, a monitor, a keyboard, a mouse, bulk fluid containers, a waste container, and related equipment. Additional staining modules or other instruments may be added to such a system to form a network with the computer functioning as a server. Alternatively, some or all of these separate components could be incorporated into the automated processing unit, making it a stand-alone instrument.
  • the automated processing unit is a microprocessor-controlled staining instrument that automatically applies chemical and biological reagents to tissue mounted on standard glass microscope slides.
  • a carousel supporting radially positioned glass slides is revolved by a stepper motor to place each slide under one of a series of reagent dispensers.
  • the automated processing unit controls dispensing, washing, mixing, and heating to optimize reaction kinetics.
  • the computer controlled automation permits use of the automated processing unit in a walk-away manner, i.e. with little manual labor.
  • automated processing unit 70 comprises a housing formed of a lower section 90 removably mounted or hinged to an upper section 92.
  • a slide carousel 94 is mounted within lower section 90 for rotation about its central axis A-A.
  • a plurality of thermal platforms 10 are mounted radially about the perimeter of carousel 94 upon which standard glass slides with tissue samples may be placed.
  • the carousel 94 is preferably constructed of stainless steel. The temperature of each slide may be individually controlled by means of various sensors and microprocessors.
  • wash dispense nozzles 96 Also housed within the automated processing unit 70 are wash dispense nozzles 96, CoverslipTM dispense nozzle 98, a fluid knife 100, a wash volume adjust nozzle 102, bar code reader mirror 104, and air vortex mixers 106.
  • the automated devices typically include a rotatable reagent carousel 108.
  • Dispensers 110 are removably mounted to a reagent tray 112 which, in turn, is adapted to engage the carousel 108.
  • Reagents may include any chemical or biological material conventionally applied to slides including nucleic acid probes or primers, polymerase, primary and secondary antibodies, digestion enzymes, pre- fixatives, post-fixatives, readout chemistry, and counterstains.
  • Reagent dispensers 110 are preferably bar code labeled 114 for identification by the computer. For each slide, a single reagent is applied and then incubated for a precise period of time in a temperature-controlled environment. In certain embodiments, compressed air jets 106 aimed along the edge of the slide cause rotation of the reagent, thereby mixing the reagents.
  • the reagent is washed off the slide using the nozzles 96. Then the remaining wash buffer volume is adjusted using the volume adjust nozzle 102. CoverslipTM solution, to inhibit evaporation, is then applied to the slide via nozzle 98. Air knife 100 divides the pool of CoverslipTM solution followed by the application of the next reagent. These steps are repeated as the carousels turn until the protocol is completed.
  • the automated processing unit 70 preferably includes its own microprocessor to which information from the host computer is downloaded.
  • the computer downloads to the microprocessor both the sequence of steps in a run program and the sensor monitoring and control logic called the "run rules" as well as the temperature parameters of the protocol.
  • Model No. DS2251T 128K from Dallas Semiconductor, Dallas, Texas, is an example of a microprocessor that can perform this function.
  • the automated processing unit 70 generally comprises about twenty thermal platforms 10, radially mounted to a carousel 94, for heating the slides and monitoring the temperature thereof, and a control electronics printed circuit board also mounted to the slide carousel 94 for monitoring the sensors 48 and controlling the heaters 46.
  • the control electronics are mounted under the rotating slide carousel 94. Information and power are transferred from the fixed instrument platform to the rotating carousel 94 via a slip ring assembly. This information includes the temperature parameters needed for heating the slides (upper and lower) communicated from the microprocessor 116 (after having been downloaded from the computer) to the control electronics as described below. If, during a run, the slide temperature is determined to be below the programmed lower limit, the thermal platform 10 heats the slide. Likewise, if the slide temperature is found to be above the upper limit, heating is stopped. A power supply of sufficient capacity to provide about eight watts per heater is provided to meet the requisite rate of temperature rise (a/k/a "ramp up").
  • Slide cooling may be likewise controlled, as described subsequently.
  • cooling platforms are mounted below the slide.
  • the cooling platforms may comprise Peltier-type thermal transducers.
  • a cooling device such as a fan, optionally may be provided if rapid cooling of the slides is required for particular applications.
  • Certain disclosed slide heating system use conduction heating and heats slides individually.
  • the system provides more accurate on-slide temperature and allows for temperature settings on a slide-by-slide basis.
  • the heater/sensor unit should have the ability to rapidly heat the useful area of the slide from 37 0 C to 95 0 C in under two minutes and to cool down over same range in under four minutes so as to permit DNA denaturation without over-denaturation and loss of cell morphology due to excess heating.
  • Certain disclosed embodiments include control electronics.
  • certain control electronics include an annular printed circuit board with the components necessary to receive temperature setpoint information from the automated processing unit's microprocessor 116 via a serial digital protocol. This information is used to maintain each heater 46 at its setpoint. Heater control may be performed in a variety of methods.
  • the heaters 46 may be individually controlled by an integrated circuit driver or individual transistors capable of switching the heater current on and off.
  • the processor may control the duty cycle of the heaters 46, as described subsequently.
  • the amount of power to the heaters 46 may be regulated by a processor so that the heaters may be performed at a percentage of total capacity (e.g., 50% of maximum heating power).
  • the control electronics functions to both monitor the temperature sensors 48 and control the heaters 46.
  • Central to control electronics is a microprocessor (which is in communication with memory) or other digital circuitry of sufficient capability to communicate with the automated processing unit' s microprocessor 116 via a serial bus, monitor the heater temperature sensors 48, and power the heaters 46 when the slide temperature needs to be raised at a particular time.
  • a microprocessor is PIC16C64A available from Microchip Technology Inc., Chandler, Ariz.
  • the program controls each heater 46 in response to the heater temperature sensor 48 when compared with the setpoint temperature (or target temperature) provided by the automated processing unit's microcontroller.
  • the microprocessor determines the setpoint temperatures in the memory to control the heaters 46.
  • the setpoint temperatures in a look-up table are received from the serial communication with the automated processing unit's microprocessor 116.
  • the control electronics microprocessor obtains the actual temperatures from the temperature sensors 48, and thereafter modifies the control of the heaters 46 based on the difference in actual temperature and setpoint temperature.
  • This control of the heaters 46 may be strictly on-off (i.e., turn the heater on if its sensor 48 temperature is below setpoint, and turn the heater off if its sensor temperature is above setpoint) or it may use proportional, integral, and/or derivative control system algorithms to provide a more controlled and accurate response.
  • the automated processing unit may be used to perform in-situ hybridization (ISH), in-situ PCR, immunohistochemistry (IHC), as well as a variety of chemical (non-biological) tissue staining techniques. Moreover, two or more of the above techniques may be employed during a single run despite their differing temperature requirements by using individual slide heating systems.
  • ISH in-situ hybridization
  • IHC immunohistochemistry
  • two or more of the above techniques may be employed during a single run despite their differing temperature requirements by using individual slide heating systems.
  • In-situ hybridization is clearly a technique that may be advantageously employed with the present invention, either alone or in combination with other techniques, since many of the steps in ISH must be carefully temperature controlled for a precise period of time. The precise amount of heat for a specific period of time is necessary to sufficiently denature DNA so that subsequent hybridization may occur without over-heating to the point where cell morphology is degraded. Different specimens may require different temperatures for denaturation depending on how the tissue was prepared and fixed. The steps of denaturation, hybridization, and posthybridization washes each have unique temperature requirements that depend on the particulars of the probe and tissue being tested. These temperature requirements can be controlled through the individualized control of heaters.
  • DNA probes require and are typically hybridized at between 30 °-55 0 C while RNA probes are typically hybridized at higher temperatures with the time for hybridization varying from 30 minutes to 24 hours depending on target copy number, probe size and specimen type.
  • Standard denaturation for cytogenetic preparations is performed at about 72 0 C for 2 minutes while for tissue sections the conditions may vary from 55 0 C to 95 0 C from 2 to 30 minutes.
  • Post hybridization wash temperatures may vary from about 37 0 C to 72 0 C for 2 minutes to 15 minutes.
  • Salt concentration may vary from 0. Ix to 2x SSC.
  • Probe detection temperatures may vary from ambient to 42°C for 2 minutes to 30 minutes.
  • ISH may be employed to detect DNA, cDNA, and high copy mRNA. It can be applied to smears, tissue, cell lines, and frozen sections. Typically, the specimen is mounted on a I"x3" glass slide.
  • ISH includes both fluorescent detection (FISH) and non-fluorescent detection (e.g. brightf ⁇ eld detection).
  • the automated processing unit also may be employed for simultaneous application of ISH and IHC staining to certain tissue sections to allow both genetic and protein abnormalities to be viewed at the same time. This may be advantageous, for example, in assaying breast tumor sections for both gene amplification and protein expression of HER-2/neu as both have been deemed to have clinical significance.
  • PCR polymerase chain reaction
  • a limitation of PCR is the need to extract the target DNA or RNA prior to amplification that precludes correlation of the molecular results with the cyto logical or histological features of the sample.
  • In situ PCR obviates that limitation by combining the cell localizing ability of ISH with the extreme sensitivity of PCR. The technique is described in U.S. Pat. No. 5,538,871 to Nuovo et. al, which is incorporated herein by reference. Sections embedded in paraffin require as a first step deparaffmization of the embedded tissue.
  • Using a thermal platform eliminates the use of harsh chemicals, such as xylene, because precisely controlled heating of individual slides allows the paraffin embedded in the tissue to melt out and float in aqueous solution where it can be rinsed away. Liquid paraffin can then be removed from the microscope slide and away from the tissue sample by passing a fluid stream, either liquid or gaseous, over the liquid paraffin.
  • a similar technique may be employed to remove embedding materials other than paraffin such as plastics although the addition of etching reagents may be required.
  • Typical in-situ Hybridization (ISH), in-situ PCR, Immunohistochemical (IHC), Histochemical (HC), or Enzymehistochemical (EHC) methods as carried out with the apparatus of this invention includes the following steps.
  • each slide will be dry heated to temperatures above 60 0 C. Following the dry heat, the slides are washed with about 7 ml of DI water leaving a residual aqueous volume of about 300 ⁇ l. The slides are then covered with about 600 ⁇ l of evaporation inhibiting liquid. The slides remain at temperatures above 60 0 C for an additional 6 minutes and are then rinsed again with about 7 ml DI water and covered with 600 ⁇ l of evaporation inhibiting liquid. The temperature of lowered to 37 0 C. The slides are deparaffinized and ready for the next phase of the indicated process. 5) Slides that are to be cell conditioned will be rinsed with about 7 ml DI water.
  • a volume-reducing fixture within the apparatus will lower the residual volume from about 300 ⁇ l to about 100 ⁇ l.
  • Using a volume-adjusting fixture within the apparatus 200 ⁇ l of cell conditioning solution will be added to the slide.
  • the slide will then receive about 600 ⁇ l of evaporation inhibiting liquid.
  • the slide temperature will be raised to the assigned temperature in a range of 37 0 C to 100 0 C, and fluid cycling will commence and be repeated every 6-8 minutes for a period of time up to 2 hours as set in the protocol. Slides are cooled to 37 0 C and rinsed with about 7 ml of APK wash solution. At this point the slides are ready for the next phase of the indicated process.
  • the appropriate reagent vessel is moved by the reagent carousel to the reagent application station.
  • a metered volume of reagent is applied to the slide.
  • the reagent liquid passes through the evaporation inhibiting liquid layer to the underlying liquid layer.
  • slides can be rinsed with about 7 ml of APK wash solution leaving a residual volume of about 300 ⁇ l of buffer.
  • the slide will then receive about 600 ⁇ l of evaporation inhibiting liquid.
  • Steps as described in steps 6 and 7 are repeated for digestive enzyme application. Selectable incubation times range from 2 min through to 32 minutes at 37 0 C.
  • the slides are rinsed with about 7 ml of 2x SSC buffer, leaving a residual volume of about 300 ⁇ l of buffer.
  • a volume reducing fixture is used to shift the volume from about 300 ⁇ l to about 100 ⁇ l. Steps as described in 6 and 7 are repeated for probe application.
  • the slide will then receive about 600 ⁇ l of evaporation inhibiting liquid.
  • slides will be rinsed with DI water and a detergent will be applied to clear the slides of the evaporation inhibiting liquid. Again the slides will be rinsed with DI water and the residual volume will be removed with the use of the volume reducing fixture. The slides will be dry heated at temperatures at or above 37 0 C until all aqueous is evaporated from the tissue, cells or smears. 8b) For in-situ PCR If process requires protein digestion, slides are rinsed with about 7 ml of APK wash solution leaving a residual volume of about 300 ⁇ l of buffer. The slide will then receive about 600 ⁇ l of evaporation inhibiting liquid. Steps as described in steps 6 and 7 are repeated for digestive enzyme application.
  • Selectable incubation times range from 2 minutes through to 32 minutes at 37 0 C.
  • the slides are rinsed with about 7 ml of DI water, leaving a residual volume of about 300 ⁇ l of buffer.
  • a volume reducing fixture is used to shift the volume from about 300 ⁇ l to about 100 ⁇ l.
  • Steps as described in step 7 are repeated for amplification reagent application.
  • Amplification reagents are formulated for delivery to 100 ⁇ l residual slide volume at temperatures at or above 37°C.
  • the slide will then receive about 600 ⁇ l of evaporation inhibiting liquid. Raise slide temperature to specified temperature in a range of 37 0 C to 95 0 C for greater that 2 minutes to start PCR reaction. Heat cycling, up to 30 cycles, will commence from 55 0 C for 1.5 minutes to 89 0 C for 45 seconds. Following In-Situ PCR the slides will be subjected to in-situ Hybridization.
  • the slides are rinsed with about 7 ml of Ix APK wash or appropriate buffer, leaving a residual volume of about 300 ⁇ l of buffer.
  • a volume reducing fixture may or may not be used to shift the volume from about 300 ⁇ l to about 100 ⁇ l.
  • the slide will then receive about 600 ⁇ l of evaporation inhibiting liquid. Steps as described in step 7 are repeated for antibody or other reagent application.
  • Selectable incubation times range from 2 minutes to 32 minutes. Selectable incubation temperatures range from 37 0 C to 95 0 C depending on whether cell conditioning or deparaffinization is required.
  • the slides are washed with Ix APK wash or appropriate buffer, and then receive about.600 ⁇ l of evaporation inhibiting liquid. Proteins, carbohydrates, and enzymes are directly labeled, as in fluorescence, or indirectly using an appropriate detection technology.
  • the slides are prepared for coverslipping with the automated clearing procedure, coverslipped, and reviewed microscopically for appropriate staining, be it DNA/RNA, protein, carbohydrate or enzyme.
  • Within-run and between run temperature control is ⁇ 1% of the target temperature and may be controlled as described previously.
  • the operator may run multiple complex ISH protocols in the same run. This includes ability to program protocols for ISH methods that run at the same denaturation temperatures.
  • the system is accessible for slide temperature calibration by the operator without tedious dismantling of the instrument.
  • the protocol changes for user- defined ISH protocols are protected by a security access.
  • the system is barcode driven for both the slide and reagent system.
  • User defined protocols allow the operator to control the temperature of all phases of the reaction except detection temperature.
  • the software includes pre-programmed optimized protocols resident in the software to allow continuous introduction of the optimized turnkey probes.

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  • Physics & Mathematics (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Optics & Photonics (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Sampling And Sample Adjustment (AREA)

Abstract

L'invention est liée au traitement automatisé de lames de microscope, au cours duquel une lame de microscope contenant un échantillon sur une face peut être placée sur une plate-forme, le côté échantillon de la lame étant tourné vers le haut. Auparavant, un fluide employé dans le traitement des lames pouvait déborder par-dessus les bords d'une lame et s'écouler jusque sous la lame, contaminant une face arrière de la lame. Les systèmes décrits ici surmontent ces difficultés en isolant une région de la lame qui coopère avec une plate-forme. Les systèmes selon l'invention peuvent comporter une plate-forme configurée en vue de recevoir une lame de microscope et un joint entourant la plate-forme. Lorsque la surface inférieure de la lame de microscope entre en contact avec le joint, un réducteur de pression peut être actionné et couplé fluidiquement à un volume délimité en partie par la plate-forme et par la surface inférieure de la lame de microscope.
PCT/US2009/068303 2008-12-17 2009-12-16 Appareil d'isolement du dessous d'une lame Ceased WO2010077975A1 (fr)

Applications Claiming Priority (2)

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US13836508P 2008-12-17 2008-12-17
US61/138,365 2008-12-17

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WO2010077975A1 true WO2010077975A1 (fr) 2010-07-08

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102011007324A1 (de) * 2011-04-13 2012-10-18 Carl Zeiss Microlmaging Gmbh Objektträgerhalter
US10634590B2 (en) 2014-03-11 2020-04-28 Emd Millipore Corporation IHC, tissue slide fluid exchange disposable and system
US11506579B2 (en) * 2013-10-04 2022-11-22 Biogenex Laboratories, Inc. Assembly for forming microchamber for inverted substrate
CN115436372A (zh) * 2022-10-12 2022-12-06 马倩 环境保护用人工智能检测装置及其检测方法

Citations (5)

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Publication number Priority date Publication date Assignee Title
US3848962A (en) * 1973-10-18 1974-11-19 Coulter Electronics Slide mounting apparatus for microscopy
US4508435A (en) * 1982-06-18 1985-04-02 Coulter Electronics, Inc. Air vacuum chuck for a microscope
US5439649A (en) * 1993-09-29 1995-08-08 Biogenex Laboratories Automated staining apparatus
US5659421A (en) * 1995-07-05 1997-08-19 Neuromedical Systems, Inc. Slide positioning and holding device
US20030211630A1 (en) * 1998-02-27 2003-11-13 Ventana Medical Systems, Inc. Automated molecular pathology apparatus having independent slide heaters

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3848962A (en) * 1973-10-18 1974-11-19 Coulter Electronics Slide mounting apparatus for microscopy
US4508435A (en) * 1982-06-18 1985-04-02 Coulter Electronics, Inc. Air vacuum chuck for a microscope
US5439649A (en) * 1993-09-29 1995-08-08 Biogenex Laboratories Automated staining apparatus
US5659421A (en) * 1995-07-05 1997-08-19 Neuromedical Systems, Inc. Slide positioning and holding device
US20030211630A1 (en) * 1998-02-27 2003-11-13 Ventana Medical Systems, Inc. Automated molecular pathology apparatus having independent slide heaters

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102011007324A1 (de) * 2011-04-13 2012-10-18 Carl Zeiss Microlmaging Gmbh Objektträgerhalter
DE102011007324B4 (de) 2011-04-13 2022-10-13 Carl Zeiss Microscopy Gmbh Objektträgerhalter
US11506579B2 (en) * 2013-10-04 2022-11-22 Biogenex Laboratories, Inc. Assembly for forming microchamber for inverted substrate
US10634590B2 (en) 2014-03-11 2020-04-28 Emd Millipore Corporation IHC, tissue slide fluid exchange disposable and system
CN115436372A (zh) * 2022-10-12 2022-12-06 马倩 环境保护用人工智能检测装置及其检测方法

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