WO2011139976A1 - Coloration de cellules avec chauffage à la vapeur et refroidissement par air - Google Patents

Coloration de cellules avec chauffage à la vapeur et refroidissement par air Download PDF

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Publication number
WO2011139976A1
WO2011139976A1 PCT/US2011/034824 US2011034824W WO2011139976A1 WO 2011139976 A1 WO2011139976 A1 WO 2011139976A1 US 2011034824 W US2011034824 W US 2011034824W WO 2011139976 A1 WO2011139976 A1 WO 2011139976A1
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WIPO (PCT)
Prior art keywords
steam
air
temperature
slide
chamber
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PCT/US2011/034824
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English (en)
Inventor
Joseph Del Rossi
Patrick Dunlop
Stephanos Michael
Stephanie Sanchez
Sean P. Schoonmaker
Ali Yetisen
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Ventana Medical Systems, Inc.
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Publication of WO2011139976A1 publication Critical patent/WO2011139976A1/fr

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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00Sampling; Preparing specimens for investigation
    • G01N1/28Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G01N1/31Apparatus therefor
    • G01N1/312Apparatus therefor for samples mounted on planar substrates
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01FMIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
    • B01F31/00Mixers with shaking, oscillating, or vibrating mechanisms
    • B01F31/20Mixing the contents of independent containers, e.g. test tubes
    • B01F31/23Mixing the contents of independent containers, e.g. test tubes by pivoting the containers about an axis
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01FMIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
    • B01F31/00Mixers with shaking, oscillating, or vibrating mechanisms
    • B01F31/65Mixers with shaking, oscillating, or vibrating mechanisms the materials to be mixed being directly submitted to a pulsating movement, e.g. by means of an oscillating piston or air column
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N2035/00346Heating or cooling arrangements
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N2035/00465Separating and mixing arrangements

Definitions

  • This application relates to cell staining technology, and in particular to automated cell staining equipment and methods.
  • Cell staining methods including immunostaining and in situ DNA analysis, are useful tools in histological diagnosis and the study of tissue morphology.
  • Immunostaining relies on the specific binding affinity of antibodies with epitopes in tissue samples, and the increasing availability of antibodies which bind specifically with unique epitopes present only in certain types of diseased cellular tissue.
  • Immunostaining requires a series of treatment steps conducted on a tissue section mounted on a glass slide to highlight by selective staining certain morphological indicators of disease states. Typical steps include pretreatment of the tissue section to reduce non-specific binding, antibody treatment and incubation, enzyme labeled secondary antibody treatment and incubation, substrate reaction with the enzyme to produce a fluorophore or chromophore highlighting areas of the tissue section having epitopes binding with the antibody, counterstaining, and the like. Each of these steps is separated by multiple rinse steps to remove unreacted residual reagent from the prior step. Incubations are conducted at elevated temperatures, usually around 40°C, and the tissue must be continuously protected from dehydration.
  • In situ DNA analysis relies upon the specific binding affinity of probes with unique nucleotide sequences in cell or tissue samples and similarly involves a series of process steps, with a variety of reagents and process temperature requirements.
  • Current cell staining technology includes the Ventana BenchMark ULTRA, which is a fully automated instrument capable of processing 30 independent samples simultaneously.
  • the BenchMark ULTRA system covers the tissue sample by continually applying the reagent until surface tension is overcome and the entire slide is covered. With this method, it takes about 600 microliters ( ⁇ ) of reagent to achieve coverage.
  • the 600 ⁇ 1 solution contains ⁇ of active ingredient and 500 ⁇ 1 of buffer included to assure complete coverage of the slide.
  • Mixing and constant interaction between the sample and the reagents is achieved by two opposing and tangential air jets which produce a mixing vortex.
  • the current method of heating for the incubation period is through conduction using an electrical heating plate.
  • a layer of oil on top of the reagents can be added to address evaporation.
  • the present apparatus and methods seek to advance and improve upon those in current use.
  • an automated cell staining apparatus comprises a chamber enclosing a microscope slide having a slide thereon, a steam heat source and an air source.
  • the steam heat source is capable of heating the chamber, microscope slide and sample with steam to a desired temperature.
  • the air source is capable of cooling the steam heat source with air for controllably reaching the desired temperature.
  • the steam heat source is capable of heating the microscope slide and sample to a desired temperature between 35°C to 85°C.
  • the apparatus is capable of maintaining the desired temperature within 1°C over a duration of testing.
  • the apparatus is capable of maintaining different points across the microscope slide within 1.25°C of the desired temperature.
  • the steam heat source is capable of heating the sample to the desired temperature within about three minutes, and more preferably, within about two minutes.
  • the steam heat source can include a flash boiler connected to a source of water and capable of heating water to steam by resistive heating.
  • the steam heat source can include a spider valve positioned upstream from the flash boiler and downstream from the source of water.
  • the apparatus can include a controller programed to heat the slide quickly using a first stage combination of steam and air to a temperature close to but less than the desired temperature followed by a second stage combination of steam and air to heat the slide to the desired temperature with minimal overshooting.
  • the air source can be controlled by causing an air valve to pulse open and closed.
  • the steam can be directed in a substantially laminar flow and to heat the slide and sample through condensation heating.
  • the slide can be positioned substantially level, and the source of steam heat can be configured to direct steam substantially parallel to a lower surface of the slide.
  • the apparatus can comprise a generally horizontal duct positioned beneath the microscope slide that is capable of guiding steam and air entering the chamber in a direction along a lower surface of the slide until the steam and air exit the duct and are caused to flow over an upper surface of the slide.
  • the chamber can be insulated and generally sealed from external surroundings.
  • an automated cell staining apparatus comprises an insulated chamber, a steam heat source, an air source, a wye valve and a controller.
  • the insulated chamber is dimensioned to enclose a microscope slide having a sample thereon and comprises a steam and air inlet in a lower surface, a duct leading upwardly from the steam and air inlet and having a horizontal portion along a lower surface of the microscope slide, a vent in an upper surface of the chamber, and a temperature sensor capable of sensing the temperature in the chamber.
  • the steam heat source is capable of supplying steam to heat the chamber, the microscope slide and the sample to desired temperatures by condensation heating, and comprises a flash boiler powered by cartridge heaters and a controllable steam valve.
  • the air source is capable of generating air at a temperature cooler than the steam, the air source comprising an air pump and a controllable air valve. Steam from the flash boiler and the controllable steam valve, and air from the air pump and the controllable air valve, are combined at the wye valve and fed to the air and steam inlet of the chamber, the resulting mixture having a temperature less than a temperature of the steam.
  • the controller is connected to the temperature sensor, the controllable steam valve and the controllable air valve.
  • the controller is programmed to cause the chamber to reach a desired set point temperature by actuating the controllable steam valve to supply more steam if a current temperature sensed by the temperature sensor is below the desired temperature and by actuating the controllable air valve to supply more air if the current temperature sensed by the temperature sensor is above the desired temperature.
  • Fig. 1 is a schematic view of a control volume that represents a chamber of the enhanced cell staining apparatus.
  • Fig. 2 is a drawing showing a representative velocity profile of a flow into the chamber.
  • Fig. 3 is another schematic control volume representing the chamber with steam and air inputs.
  • Fig. 4(a) is a schematic view showing liquid film condensation on a surface.
  • Fig. 4(b) is a schematic view showing dropwise condensation on a surface.
  • Fig. 5 is a schematic view showing various fluid flow regimes for condensation on the outer surface of a slide as the thickness of the condensation increases.
  • Fig. 6 is a perspective view showing an embodiment of a horizontal duct for positioning beneath the microscope slide.
  • Fig. 7 is a perspective view of an oscillating platform for holding a microscope slide and mixing liquids applied to the slide according to a first approach.
  • Fig. 8(a) is a second alternative mixing approach of a translating head that drags reagent across the slide through capillary action at a gap between the head and an upper surface of the slide.
  • Fig. 8(b) is a third alternative mixing approach of a shaped cap that is caused to rock on a slide, thereby causing liquid on the slide to be mixed.
  • Fig. 9 is a Pugh matrix showing weightings for various test scenarios.
  • Fig. 10 is a perspective view of the overall testing apparatus.
  • Figs. 11(a)- 11(d) are various views of a chamber within which the translating gap assembly and microscope slide are placed to isolate them from the exterior surroundings.
  • Fig. 12 is a cross-sectional view in elevation of the chamber showing paths for the steam/air mixture and its path of flow over the microscope slide.
  • Figs. 13(a)- 13(g) are various views of a translating gap head.
  • Fig. 14(a) is a front elevation view of a translating gap head in position over a microscope slide.
  • Fig. 14(b) is an enlarged cross-sectional view in elevation showing the gap between the translating gap head and the microscope slide at a point spaced away from the mixing projection.
  • Fig. 14(c) is a cross-sectional view in elevation showing the smaller gap between the mixing extension on the translating gap head and the microscope slide.
  • Fig. 15 is a perspective view showing the motion of the translating gap head relative to the microscope slide.
  • Figs. 16(a)-16(g), Figs. 17(a)-17(c), and Figs. 18(a)-18(f) are various views of an alternative embodiment of a translating gap head that provides for removal of liquid from the slide by suction as well as supply of liquid to the microscope slide.
  • Fig. 19 is a perspective view of a linear guide screw powered by a stepper motor for moving the translating gap head.
  • Figs. 20 and 21 are elevation views of the linear guide screw and translating gap head in operation.
  • Figs. 22(a)-22(c) are various views of the flash boiler.
  • Fig. 23 is a perspective view of a portion of the system showing the chamber and the air and steam inputs to the chamber.
  • Fig. 24(a) is a schematic showing the controller and the connections to various other elements of the system.
  • Fig. 24(b) is another schematic view of the controller and connections to other components of the system.
  • Fig. 25 is a system controls and integration diagram.
  • Fig. 26(a) is a graph of temperature versus time for eight different combinations of steam and air.
  • Fig. 26(b) is a table of the supporting data for Fig. 26(a).
  • Fig. 26(c) summarizes air delay values and steam delay values for the respective combinations.
  • Fig. 27(a) is a graph of temperature versus time showing the time required to reach three different set point temperatures.
  • Fig. 27(b) is a table of the supporting data for the graph of Fig. 27(a).
  • Fig. 28(a) is a perspective view of the position of sensors on a slide to test for temperature uniformity.
  • Fig. 28(b) is a graph of temperature uniformity results.
  • Described below are improved cell staining systems and methods that are automated, thermally isolated, and capable of extension into a multi-cell commercial instrument.
  • the new systems and methods offer several advantages, such as, e.g., providing faster heating, improving temperature uniformity, providing better mixing, providing evaporation control and/or reducing required reagent volumes.
  • the system performs basic functions necessary for the cell staining process.
  • the system can reach a specific set-point temperature on a microscope slide between 35-85°C and maintain that temperature with no more than a 1°C variation relative to the set-point temperature.
  • the system should also maintain temperature uniformly across the microscope slide with no more than 0.5°C variation from one portion of the slide to another. This tight tolerance ensures that the entire tissue sample is stained evenly.
  • the system should be able to reach any set-point temperature in less than 3 minutes, which is the time required by the current Benchmark ULTRA.
  • the system should eliminate evaporation of reagents from the microscope slide during the incubation period.
  • the system should completely cover the microscope slide with reagents in order to insure that the entire tissue sample is covered.
  • the system should then thoroughly remove reagents from the microscope slide.
  • the system is fully automated.
  • a conventional reagent dispensing carousel is envisioned.
  • reagent is generally applied from the top of the slide.
  • the method of heating the slide in the new system utilizes steam.
  • the presence of steam affects many design aspects, including venting, drainage, size and corrosion.
  • Removal of reagents from the slide in the new system is desirably through vacuum aspiration.
  • the system should accommodate a suction apparatus above the slide together with a passageway for waste disposal, such as through suitable tubing.
  • a multi-slide processing instrument each slide can be accommodated in an adiabatic chamber to prevent temperature influence on neighboring slides.
  • the nozzle that releases steam-air mixture to the chamber operates for long periods of time under the same conditions. There will be a transient period where the slide reaches a certain temperature and the heat transfer rate to the slide becomes steady. Therefore, there is no change in intensive or extensive properties in the control volume region. This means that the volume, mass and the total energy content must remain constant. There is no boundary work. The total mass or energy entering the system should be equal to the total mass or energy leaving the system.
  • the chamber for each slide can be illustrated as a control volume, e.g., as shown in Fig. 1.
  • thermodynamics states that energy can be transformed (changed from one form to another), but cannot be created or destroyed.
  • the difference of an amount of energy entering a system and amount of energy leaving the system has to be energy change in the system. This can be negative or positive, i.e., cooling can be accomplished by losing energy and heating can be obtained by receiving energy from the outside environment.
  • the energy balance for closed systems is expressed as:
  • the total amount of energy entering the system is equal to the total amount of energy leaving the system.
  • the chamber receives energy from the outside environment via tubing.
  • the chamber is assumed to be an ideal case, and thus it is assumed that there is no heat loss through the insulation.
  • the weights of the components of the chamber parts (Fig. 10) are obtained from CAD (Solidworks).
  • the energy can be expressed in terms of mass, specific heat and the temperature change of the system.
  • the required energy to raise each of the individual components of the chamber is calculated as follows. cp,ABS plastic ⁇ 1-2 ⁇ [4] Cp,glass ⁇ 0-84— Q— m.C p T
  • the mass flow rate can cause the temperature of the chamber to increase or decrease. If the energy input is less than the energy loss, the chamber temperature will decrease. Regulated mass flow rate determines the accuracy of the energy input. In fluid dynamics, a mixture travelling through a duct will have a zero velocity at the solid wall. Considering a no slip condition at the walls, the velocity profile develops, concentrating in the middle, as shown in Fig. 2.
  • the differential mass flow rate that flows through an area of dA c relationship can be expressed as: ⁇ — p V nor maimer oss section (2-1)
  • the mass flow rate can be expressed in terms of average values where the average velocity can be defined as:
  • ⁇ average the average velocity throughout the cross section of the nozzle
  • volume flow rate can be expressed as,
  • the velocity of the air exiting the nozzle was determined by experimental testing.
  • the nozzle consists of a circular duct, fed by a motor.
  • the experiments were performed at the exit of tubing where the air leaves into the atmosphere.
  • a pressure transducer is a capacitance device in which the measured capacitance changes when the pressure on one of its side moves its diaphragm. It indicates the pressure in inches of water, which is a unit of choice when measuring small pressure differences.
  • the experiments were performed using three different tubing lengths to note the pressure losses due to the length of the tubing. Pa
  • the nozzle inlet at the flash boiler feeds the boiler with water.
  • the weight the water dispensed from the nozzle is obtained using an analytical balance and dividing by the time to obtain the mass flow rate.
  • Average value is obtained to be 0.00127
  • the steam at 100°C is to be mixed with air at 24°C.
  • Individual temperatures and mass flow rates of the air and water are used as an input to find the mixture temperature and mass flow rate.
  • the mixing chamber is well insulated and does not involve any kind of work on the system. The kinetic and potential energies of the system are also negligible during the mixing process.
  • T ⁇ denotes steam at 100°C
  • T 2 denotes air at 24°C being mixed in the chamber.
  • the ratio of the mass flow rates of the respective T ⁇ and T 2 streams is required to obtain a desired output. The ratios are entered into the control system of the device.
  • the mixing chamber is taken as the system. It is considered as the control volume because mass crosses through the system. There are two inlets and one outlet.
  • the mass balance can be written as: dm ⁇ system
  • the equation 3.7 provides the enthalpy of the mixture when the inputs are the atmospheric air and steam at 100°C. The ratios are further incorporated into the test plan and the control system.
  • the amount of water vapor in atmospheric air supplied to the air-steam mixture can vary. It is not a fixed quantity and differs depending on the location and conditions of the weather. The amount of water in the air has to be determined theoretically in order to calibrate the device each time it starts the operation cycle. The equations are used in the testing process of the device. The most proper way is to use mass of water vapor present in a unit mass of dry air. Absolute or specific humidity (denoted as ⁇ ) can be expressed as following,
  • Dry air contains no vapor.
  • the specific humidity will increase as vapor is added.
  • the specific humidity will increase until the air cannot accommodate more moisture. At this point the air is defined as saturated air. If moisture is added to the air, it will condense.
  • the ratio of the amount of moisture the air holds (mv) to the maximum amount of moisture that the air can hold at the same temperature (mg) is called relative humidity (denoted as ⁇ ).
  • relative humidity
  • Relative humidity ranges from 0 (dry air) to 1 (saturated air).
  • the amount of moisture that dry air can hold depends on its temperature. Therefore, the device needs to be calibrated at the beginning of each operation cycle. The calibration will provide a reference point that the control system can calculate the ratio of steam to air to be mixed.
  • the enthalpy of atmospheric air is the sum of the enthalpies of dry air and water vapor:
  • the partial pressure of dry air can be determined from
  • the fluid mixture that is fed to the chamber consists of air at 24°C and steam at 100°C.
  • Air is a mixture of mainly nitrogen and oxygen and some other gasses in small amounts. Air present in the atmosphere has some water vapor and called atmospheric air. However, if air contains no water vapor, it is called dry air. In this particular case, since the atmospheric air is used, the air can be considered as a mixture of dry air and water vapor. Assuming air temperature is 24°C, dry air can be considered as an ideal gas with a constant C p value of 1.005 kJ/kg ⁇ K. As the reference temperature taken as 0°C, the enthalpy change is the following,
  • the enthalpy of water is a function of temperature only.
  • enthalpy of water vapor in air is approximately equal to the enthalpy of saturated vapor at the same temperature.
  • the enthalpy of water vapor is 2676 kJ/kg.
  • the Average C p value of water vapor at 100°C is 1.859 kJ/kg ⁇ °C.
  • the enthalpy of water vapor can be determined from the following equation.
  • the mass flow rates were modified in order to increase and decrease the enthalpy of the mixture.
  • the temperature under a specific pressure (input) can be obtained from the psychrometric chart.
  • a computer program can be written to obtain exact temperatures in order to eliminate error.
  • Film condensation The condensate wets the surface and forms a liquid film on the surface that slides down under the influence of the gravity (in our case the flow direction). The thickness of the liquid film increases in the flow direction as vapor condenses on the film as shown in Fig. 4(a).
  • Dropwise condensation The condensed vapor forms droplets on the surface instead of a continuous film, and the surface is covered by countless droplets of varying diameters as shown in Fig. 4(b).
  • ⁇ ⁇ viscosity of the liquid, - m ⁇ s
  • the condensate in an actual condensation process is cooled further to some average temperature between T sat and T s ' releasing more heat in the process.
  • Modified latent heat of vaporization is defined as:
  • the rate of the heat transfer can be expressed as
  • the properties of the liquid should be evaluated in the film temperature
  • Tf Tsat * Ts which is approximately the average temperature of the liquid.
  • hfg should be evaluated at T sat since it is not affected by the subcooling of the liquid.
  • the steam flow then fills the chamber where it creates a 100% humid atmosphere that prevents evaporation of reagents from the microscope slide.
  • One problem discovered with the horizontal heating design is non-uniform temperature distribution across the microscope slide due to turbulence in the flow as it collides with the bottom surface of the slide and curls back.
  • the second preliminary design for steam heating is a horizontal half-tubular flow guiding duct 200. This design, which is shown in Fig. 6, guides the steam flow in a laminar fashion beneath the microscope slide where condensation heating takes place. The steam flow is then guided to the top surface of the slide where it prevents evaporation.
  • the laminar flow should provide a much more uniform distribution of heat across the microscope slide.
  • a first design named the "Hula” for its dancing Hula motion, comprises an oscillating platform to which a microscope slide is attached as shown in Fig. 7. The oscillation forces the applied reagents to overcome surface tension and cover the slide while maintaining reagent and tissue interaction. Preliminary test results showed inconsistent slide coverage, dry spots and regions not covered as desired.
  • a second proposed design known as the translating gap, has a head 220 that translates across the slide 222 and causes reagent to be dragged across the slide through capillary attraction at a gap between the head and the slide, as shown in Fig. 8(a).
  • a mixing tab may be located at the center of the gap head to create turbulence within the reagents. Turbulence increases interaction between the tissue sample and the reagents.
  • the translating gap approach can effectively cover the slide with a small volume of reagent.
  • a Pugh matrix (Fig. 9) was constructed for all four design options with corresponding weights assigned to the contending design approaches. The final scoring from the Pugh matrix indicated that the horizontal method for heating along with the translating gap approach as for reagent application was the optimal design option.
  • Fig. 10 Based on the Pugh matrix results, an integrated system of components needed to perform a standard cell staining process, as shown in Fig. 10. Tissue samples being tested are fixed to a microscope slide and placed in the adiabatic chamber to be stained.
  • the major functional components include a translating gap assembly for spreading, mixing, and removing reagents.
  • a flash boiler and an air pump are used for creating an air- steam mixture to heat the chamber.
  • Two microcontrollers are used to automate the entire system.
  • the optimal speed for gap translation was determined by observing area covered and overspill as a function of speed of the translating gap head moving across the slide. Additionally it was found that minimal amounts of reagents could be used, far below ⁇ . It was determined that a suction pressure above 5 psi is adequate for proper removal of the reagents. Expected information was obtained from the preliminary testing along with vital unforeseen discoveries. From these tests it was identified that the spray foam insulation had large thermal expansion characteristics which resulted in distortions of the chamber walls.
  • Optimum chamber ventilation was determined along with increased chamber size for inner workings.
  • an insulated chamber 302 was designed.
  • the chamber is made from ABS plastic with a dual wall construction. This dual wall construction was achieved by designing an inner shell 304 and outer shell 306, which allows for the insertion of an insulating material 308, as is shown in
  • high density polystyrene foam insulation was selected because of its operating temperature range and ability to be easily cut.
  • the chamber was designed with a flow guiding duct 308.
  • the flow guiding duct 308 directs the incoming steam/air mixture in a laminar fashion beneath the microscope slide.
  • the slide is heated through condensation heating which is kept uniform by the laminar flow, Fig. 12.
  • the duct then directs the flow above the microscope slide where the steam/air mixture creates a 100% humid atmosphere which prevents the reagents from evaporating off of the microscope slide and tissue sample.
  • the mixture is then guided to a vent 310 in the lid 312 of the chamber where it exits to the surrounding atmosphere.
  • weather stripping 314 and sealant was installed between the chamber lid interface and all chamber inlets and outlets, creating an air tight seal.
  • the inner chamber is made accessible through the hinged lid 312 which can be secured tightly with two accompanying latches 316, shown in Fig. 11(a).
  • the microscope slide is made removable with a microscope slide clip 318 and raised guides.
  • a drain hole 320 in the lower duct allows for drainage of any pooled excess condensation.
  • the translating gap head works on the principle of capillary attraction. After reagents are dispensed on to the microscope slide 402, the translating gap head makes contact with the reagent while maintaining a physical gap of about 0.003 inch between the surface of the translating gap head 400 and the top surface of the microscope slide, as shown in Fig. 14(a). The translating gap head then moves laterally back and forth across the slide (Fig. 15), dragging along with it the reagent through capillary attraction, which effectively covers the entire slide with reagent. Using this method, reagent volumes as low as 25 micro liters are sufficient for complete coverage of the microscope slide.
  • One or more mixing extensions 404 or projections, as shown, e.g., in Figs. 13(b), 13(f) and 13(g) can be added to the bottom surface of the translating head.
  • the mixing extension can be positioned even closer to the slide (e.g., by a gap G2 of about 80 microns, Fig. 14(c)) than the rest of the translating head (i.e., by a gap Gl of about 400 microns, Fig. 14(b)).
  • the mixing extension is typically narrower than the width of the translating head. In one embodiment, the mixing extension is about 25% of the width of the translating head.
  • liquid L on the slide at a level above the first gap will be pushed by the approaching mixing extension, causing some of it to deflect laterally.
  • the deflected liquid seeks a path of least resistance and tends to flow laterally along the advancing head until it encounters a part of its leading edge that is separated from the slide by a greater gap.
  • the mixing extension promotes lateral movement and mixing of the liquid(s) on the slide even when the primary motion of the head is translation in the axial direction.
  • Small holes 406 in the translating gap head's contact surface allow for the removal of the reagents from the microscope slide by means of vacuum aspiration.
  • suction is applied to the translating gap head as it moves laterally back and forth across the microscope slide to effectively remove all reagents.
  • these embodiments include passageways for providing reagents and/or rinse buffers from the translating gap head.
  • a head 800 includes a cap 820 that fits over a base 822.
  • Fig. 16(b) is a top plan view of the head 800 showing three rinse inlet ports 824.
  • Fig. 16(c) is an elevation view in section of the head 800 that shows the rinse inlet ports 824 in the cap 820 and passages 806 in the base 822 through which vacuum is applied.
  • Fig. 16(d) is a section view in elevation showing passageways 826 for conveying the rinse liquid to the slide.
  • the passageways are configured in the spaces between the cap 820 and the base 822.
  • Fig. 16(e) is a front elevation view of a magnified portion of the head 800. Specifically, Fig. 16(e) shows a lateral guide surface 830, a rail surface 812 and a lower surface of the head 805 spaced about 50 microns above the rail surface 812.
  • Fig. 16(f) is a perspective view similar to Fig. 16(a), except with a portion of the head 800 cut away to show the arrangement of the passages 806, the rinse liquid passageways 826 and a vacuum port 810.
  • Fig. 16(g) is another perspective cutaway view of the head 800.
  • Fig. 17(a) is a bottom plan view of the body 822 showing the array of vacuum inlets and the rail surfaces 812.
  • Fig. 17(b) is a front elevation view of the body 822.
  • Fig. 17(c) is an elevation view in section showing the intersection between the passages 806 and the vacuum port 810.
  • Fig. 18(a) is a perspective view of the cap 820.
  • Fig. 18(b) is a plan view of the bottom side of the cap showing the rinse inlet ports 824, as well as rinse bleed slots 830 and distribution channels 832 that lead from the rinse inlet ports.
  • Fig. 18(c) is sectioned side elevation view.
  • Fig. 18(d) is a sectioned front elevation view showing the rinse inlet ports 824 and one of the bleed slots 830.
  • the bleed slot 830 for center rinse inlet port 824 is shown in a magnified elevation view in Fig. 18(e).
  • Fig. 18(f) is a perspective view showing an underside of the cap 820.
  • the lateral back and forth motion of the translating gap head is achieved through a screw-driven linear slide 550 which is powered by a two- phase stepper motor 502.
  • the screw-driven linear guide was custom machined to fit the compact space of the interior cell staining chamber 302.
  • the vacuum aspiration required for the removal of the reagents was achieved through an electrical air pump (compressor) 518 that contains both an outlet for positive pressure as well as an inlet for suction.
  • Tubing is connected to a threaded barb valve in the back of the translating gap head mount. This tubing then exits the chamber 302 where it is passes through a solenoid air valve 608 which controls the suction.
  • the tubing is then connected to a bottle 520 that allows for the collection of exhausted reagents.
  • the bottle also contains another port where tubing connects the bottle to the suction port of the air pump 522.
  • An interconnecting air actuator 504 between the translating gap head 400 and the screw-driven linear slide provides up and down motion to the translating gap head, as shown in Figure 20. This actuator is controlled by an air valve 524 that is attached to the air pump.
  • the actuator serves two purposes. First, it allows for the removal of the microscope slide from the chamber by lifting the translating gap head vertically away from the microscope slide. The linear guide then moves the translating gap head horizontally, allowing enough room for microscope slide removal.
  • the second function the actuator 504 performs is keeping the tight tolerance of 0.003 of an inch between the contact surface of the translating gap head and the top surface of the microscope slide (Fig. 21).
  • the actuator 504 provides constant pressure to the rail surfaces 412 of the translating gap head which are in contact with the rail surface 414 of the inner chamber (Fig. 14(a)).
  • the rail surface of the inner chamber is the same surface the microscope slide rests on.
  • the translating gap head is designed so that as long as there is contact between its rail surfaces and the inner chamber, the proper gap tolerance between the head and microscope slide is maintained.
  • heating is required.
  • a particular target temperature between a range of 35-85 °C is required.
  • steam could be used as a heating element due to the fact that it will prevent evaporation of reagents from the microscope slide during the heating phase. Steam is also a much faster method of heating than through conduction.
  • Inputting steam at 100°C into a cell staining chamber to reach a target temperature can be achieved by a feedback control system.
  • This method of temperature control often leads to temperature overshooting and is time consuming when changing from one temperature to another.
  • steam is mixed with air to reach a target temperature before entering the cell staining chamber, thus effectively adding an element of control to the thermal process.
  • a feedback controller in not necessary with the application of mixing steam and air. For example, the steam air mixture at the target temperature can simply be continually pumped into the system to maintain an equilibrium temperature.
  • Equation 6 represents the target temperature as a function of the mass ratio of both steam and air.
  • the heating element of the cell staining chamber includes a system that mixes steam with air which then enters the chamber to achieve a target temperature.
  • the system first feeds small amounts of water from a pressurized bottle 630 by means of a spider valve 602 into a flash boiler 600.
  • the spider valve is a water valve that is capable of delivering volumes of water as small as a few picoliters.
  • the flash boiler 600 is a custom made resistive heating devise that instantaneously turns water in to steam, as shown in Figs. 22(a)-22(c).
  • the flash boiler is made from a single piece of aluminum that has a cylindrical channel (bore) through its center with an inlet for water and an exit for steam.
  • the flash boiler 600 is heated by two Hi-Temp Cartridge heaters that are inserted into the front face of the aluminum block on both sides of the steam channel. These cartridge heaters are controlled by an electric temperature controller which has a temperature gage and a thermocouple that is also inserted into the front face of the aluminum block.
  • the flash boiler is insulated with high temperature fiberglass board. After the input water changes phase into steam, it then travels through tubing where it then enters a "wye" mixing valve 604, shown in Figure 23. Air of a known temperature and quantity also enters the "wye” mixing valve through a solenoid air valve 606 which is feed by the air pump. The steam/air mixture, now at the target temperature, enters the chamber 302. The mixture travels through the chamber and heats all internal elements as well as the microscope slide containing the sample to be tested while affectively controlling the evaporation of the reagents from the microscope slide.
  • An iPad is programmed using the Wiring language, which is essentially C++ with a few simplifications.
  • An iPad Duemilanova mainboard microcontroller 700 was used for this project to automate the entire system. It was chosen for reasons of low cost, small component sizes, simple open source coding language, and ease of operation.
  • the PC which acts as the "brain" of the system, is programmed to control other components that handle specific tasks.
  • five mechanical relays are used to control the five valves (four air valves and one water valve).
  • a relay is an electrically operated switch that uses an electromagnet to operate the switching mechanism. It is placed in the circuit of a component that is being powered by a separate power source, and allows current to flow when it is closed.
  • the electrician is programmed to send a voltage output to the relay, which triggers the switch to open or close. This is the mechanism by which all five valves in the system are accurately controlled.
  • the stepper motor driver 503 is another component that is controlled by the chicken, and is used for precise control over the 4-wire bi-polar stepper motor 502.
  • the motor 502 requires 24V from a power supply, and is connected to the driver 503.
  • the driver 503 is then connected to the chicken output.
  • the chicken is programmed to output SV to the driver to make the motor rotate either clockwise or counter-clockwise in a set amount of rotations per second. This is the mechanism moves the translating gap head assembly back and forth along the linear screw guide 500.
  • the system is divided into two parts; heating the chamber and spreading/removing reagents.
  • Each part is being controlled by its own PC microcontroller, so that two separate programs can be running
  • the first chicken chip 700 that controls the heating aspect has outputs to two valves, one for air (606) and one for steam (602), and an LCD display 702.
  • the functional requirements for heating state that the chamber must be heated to any temperature between 35-85°C ( ⁇ 1°C) and remain steady. It must also reach this set-point temperature in three minutes or less.
  • steam heating as a constraint, it was decided that an air-steam combination would be the best way to maintain temperatures below 100°C. This is why both an air and water valve are necessary for the heating aspect of the design.
  • a two stage air-steam combination program with feedback was implemented.
  • Air and steam combinations are made by controlling the pulsing rate of the air valve and water valve (that feeds into the flash boiler to produce steam) individually.
  • the combinations were made to be able to heat quickly at first (mostly steam), and then slower as the chamber temperature approaches the set-point temperature.
  • the exact valve pulsing rates to achieve these optimal combinations were determined during testing.
  • When the chamber finally reaches the desired temperature no heat is added. If the chamber goes above the desired temperature, air is used to cool it down.
  • a temperature sensor 706 mounted inside the chamber is the mechanism used for feedback.
  • the code is written with the set-point temperature as a user input. It reads in the temperature from the sensor, calculates how far off it is from the set-point, determines which range the system currently is in, and activates the appropriate air-steam combination.
  • the LCD screen 702 displays the real time chamber temperature.
  • the second chicken chip 710 (Fig. 24(b)) for the reagent spreading and removal aspect of the design, has outputs that control two air actuator valves, one air valve for vacuum suction, and the driver 503 that runs the stepper motor 502.
  • the air actuator works by two air input ports. Air into one port raises the translating gap head. Air into the second port lowers it.
  • the second chicken chip 710 is programmed to lower the translating gap head into position so it can spread, mix and vacuum the reagents from the slide. It then raises the translating gap head so that it can pass above the slide clamp and return to its inactive position.
  • the air valve for suction stops the flow of air being sucked into the pump.
  • the second chicken chip 710 opens the valve when the translating gap head is moving back and forth across the slide so that it can suck the reagents up at the end of the process.
  • the air suction tube runs from the translating gap head through a collecting bottle for the reagents, into the valve, and then to the pump inlet.
  • the 2-phase motor 502 is the last component controlled by the second chicken chip 710, through intermediary action of the stepper motor driver.
  • Both of them will output to control a panel of LEDs that are used to inform the viewer which stage the system is in. Also, an LED will be used to indicate when the chamber has reached the set-point temperature within ⁇ 1°C.
  • Figs. 24(a) and 24(b) are circuit diagrams
  • Fig. 25 is a systems control and integration diagram.
  • High density Styrofoam Insulation was cut with a hot wire CNC foam cutter and was then inserted between the inner and outer shells of the adiabatic chamber. Silicon and weather stripping was then applied to all seams of the inner and outer shells, creating an air tight seal.
  • the translating gap assembly was then placed in position and secured to the inner chamber wall with screws.
  • This chamber assembly was then attached with screws to a custom fabricated sheet metal mount. This mount was then attached to a 2'X2'X1.5" hollow steel platform. All components such as air valves, air compressor, water bottles, pressure gages, etc., were secured to the steel platform with screws. Tubing and wires were then attached to all corresponding components and secured with zip ties. Verification and Validation
  • the chamber should be heated to any desired set-point temperature between 35-85°C in less than three minutes and maintained within +1°C.
  • stage 1 the stage 1 combination that is called on get the chamber temperature close to the set-point.
  • stage 2 the stage 2 combination that is chosen on get the chamber temperature close to the set-point.
  • a code can be written to create two temperature ranges below the user-defined input temperature. The first range covers all temperatures up to 10 degrees under the set-point. For this stage, the fast heating combination (Combo 1) is used. In the range from 10 degrees below set-point up to set-point temperature (stage 2), a second combination would be used. Combination 1 would be constant for any input temperature.
  • stage 2 the fast heating combination
  • This type of 2- stage feedback system provides the ability to meet two functional requirements. It combines the benefits of fast heating to meet time constraints and slower heating to reach and maintain set-point temperature without overshoot. When the temperature is finally met, the heating becomes inactive, and the insulation holds the temperature steady. If there is slight overshoot, air is blown into the chamber to cool it down. Any slight drop below set-point and the PC activates stage 2 heating to slowly bring it back to set-point.
  • Combination 1 could reach 80°C within 1 minute, so this was chosen as the stage 1 combination in the feedback code.
  • the stage 2 combination is chosen based on its ability to reach the user-defined set-point temperature. For example, combination 7 can reach 60°C within 3 minutes. Combination 8 can only reach 50°C within 3 minutes. Therefore, if it is desired to reach a temperature between 50-60°C, Combination 7 is chosen as the stage 2 combo. Using this logic, a single stage 2 combination was chosen for each of the five temperature ranges (35-50, 50- 60, 60-70, 70-80, 80-85) between 35-85°C.
  • a test slide 402 was divided up into a grid for the placement of 6 temperature sensors. Due to limited number of analog inputs on the PC, six was the maximum amount of sensors that could be read at once. For that reason, it was decided that two rows of three sensors (Fig. 28(a)) that run the length of the slide would provide enough data to determine how the uniformity was being affected. Based on the chamber design of a flow guiding duct beneath the slide, it was expected that the slide would heat at the end nearest the steam entrance first, and then become more uniform as the chamber reaches equilibrium.
  • the system After applying 100 microliters of food coloring (to represent a staining reagent) to the center of the slide, the system was capable of spreading the dye to achieve full coverage of the slide. Testing showed that within a certain range, varying the speed of the gap head did not have a significant effect on spreading. The speeds did however have a greater effect on the vacuuming/removing reagents phase. These tests showed that the slower the speed, the more effective the gap head is at removing reagents. Based on these tests, the program was written to use the slowest speed that maintained full spreading and mixing.
  • the system was able to spread the reagents to achieve full slide coverage. It kept the head moving during the entire test to accommodate the mixing requirement, and then successfully vacuumed all of the reagents from the surface to conclude the test.
  • the cell staining system as optimized by a steam- air mixture for heating and translating gap for reagent spreading, demonstrates that this design is a viable solution for the next generation cell staining system.
  • translating gap proved to be an effective way to spread, mix, and remove reagents.
  • This method there is a significant reduction in reagent volumes required to cover the entire surface of the slide.
  • the novel idea of steam heating proved effective in decreasing test times by speeding up the heating process.
  • the 100% humidity created by steam eliminates reagent evaporation at high temperatures. Without the threat of evaporation, there is no longer a need to cover the tissue sample and reagent with a protective oil layer. By eliminating the need for oil, we reduce the cost of waste treatment processes associated with bio wastes.
  • the uniformity test results showed that the temperature distribution across the width of the slide had very little variation.
  • the temperature uniformity down the length the slide was shown to be within 1.25°C at chamber equilibrium, but varied by as much as 20.44°C during the transition phase.
  • a repeatable trend showed that the temperature sensors nearest the steam-air input reached values much higher than those placed farther down the slide. This result is due to the slide placement in close proximity to the steam-air input.
  • Turbulence within the flow is creating stagnant concentrations of hot steam near the inlet. This non-uniform temperature distribution could be improved simply by placing the slide farther from the steam-air input. The location needs to be in the region where the flow is laminar through the duct.
  • a cap shaped to contact and spread liquid on the slide can be used instead of the translating head shown in Figs. 8(a) and 15.
  • a cap 201 may have a curved surface
  • the cap 201 can be caused to rock back and forth to spread liquid reagent 40 on the slide 60, without requiring the cap 201 to translate back and forth.
  • the rocking action of the cap 201 shifts the meniscus formed in the liquid reagent 40 on the slide 60 back and forth.
  • the rails 202 are each about 50 microns in height.
  • the slide 60 may have a bar code identifier, such as a bar code label 100 as shown in Fig. 8(b).
  • a production apparatus handling multiple slides can employ the translating gap approach or the rocking cap approach, among others, to accomplish mixing, in conjunction with the air quenched steam heating described above.
  • a reaction chamber is defined by at least the working area of the slide surface and structural elements positioned to the sides of and above the slide. There may also be another structural element positioned below the working surface of the slide.
  • the reaction chamber is constructed similar to the insulated chamber 302 to minimize loss of heat from within the space where the steam is being applied.

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Abstract

Appareil automatisé de coloration de cellules comprenant une chambre qui contient une lame de microscope portant un échantillon, une source de vapeur chaude et une source d'air. La source de vapeur chaude permet de chauffer la chambre, la lame de microscope et l'échantillon à la température souhaitée. La source d'air permet de refroidir la vapeur à la température voulue de manière contrôlée. Dans certains modes de réalisation, on trouve un capteur de température et un contrôleur programmé destinés à amener la chambre à la température conforme à la valeur de consigne au moyen d'air et/ou de vapeur.
PCT/US2011/034824 2010-05-04 2011-05-02 Coloration de cellules avec chauffage à la vapeur et refroidissement par air WO2011139976A1 (fr)

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US33132910P 2010-05-04 2010-05-04
US61/331,329 2010-05-04
US201161469680P 2011-03-30 2011-03-30
US61/469,680 2011-03-30

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019092269A1 (fr) 2017-11-13 2019-05-16 F. Hoffmann-La Roche Ag Dispositifs d'analyse d'échantillon utilisant l'épitachophorèse
WO2020074742A1 (fr) 2018-10-12 2020-04-16 F. Hoffmann-La Roche Ag Procédés de détection pour l'automatisation de flux de travail d'épitachophorèse
WO2020229437A1 (fr) 2019-05-14 2020-11-19 F. Hoffmann-La Roche Ag Dispositifs et procédés d'analyse d'échantillons
EP3988919A4 (fr) * 2019-10-30 2022-08-03 Sakura Seiki Co., Ltd. Dispositif de traitement de pièce de tissu

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5722566A (en) * 1996-03-11 1998-03-03 Ideal Ideas, Inc. Double action water gun
US5746009A (en) * 1995-04-21 1998-05-05 Voith Sulzer Papiermaschinen Gmbh Temperature control in a paper machine dryer
US20050118725A1 (en) * 1998-09-03 2005-06-02 Ventana Medical Systems, Inc. Automated immunohistochemical and in situ hybridization assay formulations
US20060219263A1 (en) * 2005-01-18 2006-10-05 Max Friedheim System and method for cleaning, disinfection, sterilization, and decontamination
US20100068757A1 (en) * 2008-08-29 2010-03-18 Angros Lee H In situ heat induced antigen recovery and staining apparatus and method

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5746009A (en) * 1995-04-21 1998-05-05 Voith Sulzer Papiermaschinen Gmbh Temperature control in a paper machine dryer
US5722566A (en) * 1996-03-11 1998-03-03 Ideal Ideas, Inc. Double action water gun
US20050118725A1 (en) * 1998-09-03 2005-06-02 Ventana Medical Systems, Inc. Automated immunohistochemical and in situ hybridization assay formulations
US20060219263A1 (en) * 2005-01-18 2006-10-05 Max Friedheim System and method for cleaning, disinfection, sterilization, and decontamination
US20100068757A1 (en) * 2008-08-29 2010-03-18 Angros Lee H In situ heat induced antigen recovery and staining apparatus and method

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019092269A1 (fr) 2017-11-13 2019-05-16 F. Hoffmann-La Roche Ag Dispositifs d'analyse d'échantillon utilisant l'épitachophorèse
WO2020074742A1 (fr) 2018-10-12 2020-04-16 F. Hoffmann-La Roche Ag Procédés de détection pour l'automatisation de flux de travail d'épitachophorèse
WO2020229437A1 (fr) 2019-05-14 2020-11-19 F. Hoffmann-La Roche Ag Dispositifs et procédés d'analyse d'échantillons
EP3988919A4 (fr) * 2019-10-30 2022-08-03 Sakura Seiki Co., Ltd. Dispositif de traitement de pièce de tissu

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