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WO2011077370A2 - Dispositif et procédé pour déposer automatiquement des réactifs sur des échantillons biologiques avec une précision élevée - Google Patents

Dispositif et procédé pour déposer automatiquement des réactifs sur des échantillons biologiques avec une précision élevée Download PDF

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Publication number
WO2011077370A2
WO2011077370A2 PCT/IB2010/055978 IB2010055978W WO2011077370A2 WO 2011077370 A2 WO2011077370 A2 WO 2011077370A2 IB 2010055978 W IB2010055978 W IB 2010055978W WO 2011077370 A2 WO2011077370 A2 WO 2011077370A2
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WO
WIPO (PCT)
Prior art keywords
reagents
image
supports
deposition
specimens
Prior art date
Application number
PCT/IB2010/055978
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English (en)
Other versions
WO2011077370A3 (fr
Inventor
Sergio Cometto
Pierluigi Buttu
Original Assignee
Intelsint S.R.L.
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Intelsint S.R.L. filed Critical Intelsint S.R.L.
Publication of WO2011077370A2 publication Critical patent/WO2011077370A2/fr
Publication of WO2011077370A3 publication Critical patent/WO2011077370A3/fr

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Classifications

    • 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
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G01N1/31—Apparatus therefor
    • G01N1/312—Apparatus therefor for samples mounted on planar substrates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
    • G01N35/1009—Characterised by arrangements for controlling the aspiration or dispense of liquids
    • G01N35/1011—Control of the position or alignment of the transfer device
    • 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
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G01N1/31—Apparatus therefor
    • G01N2001/317—Apparatus therefor spraying liquids onto surfaces
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
    • G01N2035/1027—General features of the devices
    • G01N2035/1034—Transferring microquantities of liquid
    • G01N2035/1041—Ink-jet like dispensers

Definitions

  • the present invention refers to a device and method for the automatic, high- precision and high-accuracy deposition of reagents on biological specimens.
  • Biological specimens both human and animal, such as paraffin-embedded or frozen tissue sections, cell smears, cytocentrifuge smears, needle aspirated biopsies or biopsies using other procedures, cell cultures, etc., are placed on transparent supports of different natures, such as optical microscope slides, and are naturally diaphanous. Thus, they are processed by means of the application of various reagents in order to reveal the different tissue and cell components.
  • sample coloring The application of a sequence of different reagents is generically called sample coloring. Colorings are divided into four categories: routine coloring, comprising most of the samples, special coloring, immunohistochemical coloring; coloring by means of the in situ hybridization technique.
  • the used reagents include substances that are able to generate or to improve the contrast of the image; in the case of routine and special coloring these substances are common chemical substances; in the field of the immunohistochemical research these substances are antibodies that bind to particular antigens; nucleotides that are able to bind to certain nucleotide sequences of specific genes are instead used for the in situ hybridization.
  • reagents are applied on the whole support where the specimen are placed, namely the reagents are placed on the support without considering the real position, thickness, shape and dimension of the biological specimen to be treated.
  • the best available instruments allow to deliver minimum volumes of reagents of about 50 microliters, in predetermined macro areas of the slide. Reagents are distributed on the specimen in an irregular way, without ensuring an adequate or regular covering of the latter. For such reasons, the delivered volumes remarkably exceed the ideal volumes that would be necessary to bring about the reaction.
  • a coloring performed according to this method also implies a certain extent of useless slide (specimen support) fouling.
  • a technical problem to be solved is thus the reduction of reagent consumption, since, according to the current techniques, reagents are wasted because more reagents than necessary are delivered in order to avoid or to reduce the problems set forth above.
  • Routine colorings involve the use of instruments exploiting the dip and dunk coloring technique, wherein the frames containing the slides , vertically placed, with the samples to be colored are introduced and immersed in sequence in a series of basins, each one of them containing a different reagent.
  • the reagents are directly applied on each slide, which are therefore treated one by one and not all together as in the dip and dunk systems.
  • all the aforementioned four coloring categories can be performed, but such method is applied above all for the immunohistochemistry and for the in situ hybridization, because they need more time and more complex instruments.
  • Such technique has the advantage of totally removing any contamination risk of both the sample and the reagent, and of reducing the quantity of reagent to be used but, as set forth above, the amount of used reagents should always be overabundant, in order to avoid other problems such as an insufficient coloring or homogeneity.
  • Immunocoloring depends on the specific binding affinity between antibodies and epitopes that are present in cells and tissues. Such binding affinity is used to identify specific structures and molecules that are useful for the diagnosis. Immunicoloring requires a series of treatment steps performed on a tissue section placed on a slide in order to reveal, by means of selective coloring, some morphological indicator of the illnesses.
  • a typical immunohistochemical coloring comprises the following steps:
  • Analysis by means of in situ hybridization depend on the affinity of the specific binding of the nucleotide probes with a determined sequence of nucleotides in the cell or tissue samples, and, similarly to immunohistochemistry, it comprises a series of steps with a variety of reagents and different incubation temperatures.
  • Such systems include an apparatus for depositing the reagents for the biological reactions, as shown in U.S. Pat. No. 5,595,707.
  • Such apparatus is controlled by computer, whose memory contains data relating to the reagents, such as serial number, product code (type of reagent), package size.
  • One of the characteristics of these system is the possibility to apply predetermined quantities of fluid on the slide, as described in U.S. Pat. No. 5,232,664.
  • the slides are arranged in horizontal position on a grid, and the reagents are distributed on the slides by means of one or more nozzles installed on a movable arm.
  • Minimum volumes of reagent of about 50 microliters are delivered in predetermined macro areas of the slide.
  • Reagents are distributed on the specimen in a non homogeneous way, without guaranteeing an adequate or regular covering of the latter.
  • the delivered volumes remarkably exceed the ideal volume that is necessary to bring about the chemical and biological reactions.
  • Such method also implies a certain extent of useless slide (specimen support) fouling.
  • the distributed volumes of reagent may be decided according to the dimensions of the specimen, since small specimens need smaller volumes of reagent with respect to larger specimen.
  • the current instruments allow to vary the distributed volumes of reagents according to discrete scales of the type below.
  • the aim of the present invention is to provide a device for the automatic deposition of reagents on biological specimens (coloring, as defined above) to be analyzed by a microscope, suitable to solve the problems set forth above, especially regarding the optimization of the usage of the reagents, and the precision, the accuracy and the cleanness of the performed colorings.
  • the present invention refers to a device for the automatic coloring of histological, cytological, haematological slides, that can be used for any coloring and specifically for the histochemical, immunohistochemical and in situ hybridization.
  • the device unlike what happens with the existing techniques where the reagents are placed on the support without considering the real position, the shape and the dimension of the biological specimen to be treated, is able to deposit the reagents only on the areas occupied by the specimens themselves.
  • the object of the present invention is a device for the automatic deposition of reagents on one or more biological specimens to be colored, such specimens being placed on transparent supports, the device comprising: means for the acquisition of 2D maps of the spatial positions of said biological specimens to be colored, means for depositing the reagents only on said spatial positions, on the basis of the information contained of said 2D maps.
  • a further aim of the invention is to provide a method for the automatic deposition of reagents on biological specimens suitable to optimize the consumption of such reagents.
  • a further aim of the invention is to provide a device suitable to guarantee the quality control of the coloring.
  • FIGS 1A, 1 B, 1C, 1 D show examples of specimen deposition on transparent supports
  • figure 2 shows a first example of diagram of the steps of the method that is object of the invention
  • figures 3 and 5 respectively show block diagrams of the steps A and B of the method of figure 2;
  • figure 4 shows a block diagram of the image acquisition system of fig. 3;
  • figure 6 shows a second example of diagram of the steps of the method that is object of the invention;
  • figure 7 shows an example of block diagram of the system that is object of the invention.
  • FIGS. 8 and 9 show two embodiments of the source-detector system of fig.
  • figure 10 shows an embodiment of the device that is object of the invention
  • figures 11 A, 12A show examples of images of biological specimens deposited on transparent supports
  • FIGS 11B, 12B show processing examples of the images 11 A, 12A, in order to respectively extract the profile of the specimens and the binary maps of the specimens to be colored.
  • the system according to the present invention allows to precisely and accurately dose the reagents in relation to the position, the shape and the dimension of the specimen to be analyzed, the latter being deposited on a transparent support, for example a slide.
  • the reagent is deposited only on the area of the slide that is occupied by the specimen, namely only on the specimen, with a dosing precision that can reach the magnitude of picoliters (pL), and with a positioning accuracy that can reach the magnitude of micrometers (pm).
  • the present invention also allows to vary the dosing of the reagents, also in relation to the density and the thickness of the specimen.
  • a further advantage of such method lies in that it allows to dose and to deposit the reagents also on specific components of the specimen, for example only on a cell nucleus or on specifically important portions.
  • two-time system or separate step system
  • real time system real time system
  • a method for the automatic deposition of reagents for biological specimens according to the present invention is articulated into two steps: a STEP A for the acquisition of the 2D map of the specimen to be colored and a STEP B during which the reagents are deposited only on the specimen, according to the information contained in the map obtained during the STEP A (fig. 2).
  • the execution of the two steps is separate both in space, namely there are two different systems, each one executing a step, and in time, namely first the acquisition of the whole map is completed and then the deposition of the reagents on the specimen is performed.
  • the two systems may be separate or may coexist within a single system.
  • a detailed embodiment will be set forth below. In the following, embodiments will be described with the help of functional block diagrams, whose realization is possible for the person skilled in the art.
  • Fig. 3 shows a block diagram of a system implementing the STEP A.
  • the system is formed by different input devices (on the left), a data processing unit and different actuation devices (on the right).
  • the processing unit 34 From the information acquired by the image acquisition system 31 , by the spatial position acquisition system 32, and by the other sensors that are present (e.g. limit stop, alarms, etc..) 33, the processing unit 34 synthesizes and sends the control signals to the different actuators relating to the positioning system 35, to the illumination system 36 and to other possible ancillary systems 37.
  • the image acquisition system will be correctly positioned with respect to the specimen support (the slide) in order to allow the image acquisition with a standardized relative position, illumination and exposition.
  • a metric on the image plane which will be used as a reference for calculating the position of the specimen on the slide.
  • the result is a bidimensional map (2D MAP) 38 of the specimen that is correlated to a known reference system integral with the support of the specimen itself.
  • the image acquisition system 31 is formed by a digital camera based on a CCD or CMOS sensor having appropriate resolution.
  • the system is equipped with appropriate optics and an automatic focusing system.
  • Such system allows an automatic digital image acquisition of the interesting specimen, lit by means of standard illumination techniques.
  • Various standard transmitted light illumination techniques of the specimen may be used to form an image suitable to be caught by the camera.
  • FIG. 4 An example of block diagram of the image acquisition system is shown in fig. 4, and comprises an optical system 41 , a signal conditioning block 42, and an extraction block of the image characteristics 43.
  • the acquired images, stored on appropriate supports are processed in order to obtain the desired characteristics (spatial position acquisition system 32) with appropriate image processing algorithms.
  • appropriate supports e.g. ram memory, flash memory, hard disk
  • the area of the slide occupied by the biological specimens to be colored and/or their boundaries are interesting characteristics.
  • Fig. 11 A shows an image in shades of grey of real diaphanous specimens on a slide
  • Fig. 11 B shows the results of an algorithm performing a pre- filtering of the image in order to increase the signal/noise ratio, e.g. by means of a moving average filter by rows and by columns on the pixels, followed by a boundary recognition algorithm.
  • Said algorithms may be of the type per se known.
  • Fig. 12A shows the same image as in Fig. 11 A
  • Fig. 12B shows the same binary map obtained from the image using a segmentation algorithm by means of binarization.
  • a one (black) or a zero (white) is assigned to each pixel of the original image if the pixel should be colored or not, respectively.
  • the image binarization is obtained from the original image, appropriately filtered by means of the aforementioned techniques for reducing its noise, by means of a threshold method.
  • the threshold is calculated from the average of the pixels of an appropriate area of the image of a reference slide, on which no specimen is present, acquired in standard operating conditions (luminosity, focal distance, exposition, etc.)
  • Figures 11 A and 12A and their respective 11 B and 12B show that, although it can be difficult recognizing the real borders of the regions of the slide occupied by the biological specimens, because of the weak contrast that is typical of the diaphanous specimens deposited on transparent supports and of the possible presence of dust or other contaminating materials, the used algorithms succeed in their task and give satisfactory results.
  • Fig. 5 shows a block diagram of a system implementing the STEP B.
  • the data processing unit 34 starting from the information contained in the 2D maps stored in the memory, processed during the step A by the system in fig. 3 (blocks 38 and 39), synthesizes the control signals to be sent to the positioning system 35 of the image acquisition system of the slides, to the reagent deposition system 51 and to possible additional systems 52.
  • the reagent deposition system 51 is based on a print head of the inkjet type with piezoelectric technology.
  • Devices of such type that can be used also for depositing biological material of various nature, are already used in other applications. See for example the solutions proposed by Arrayjet Ltd, which is leader in the production of high-quality microarray by means of the inkjet technology.
  • the print head produced by Xaar, specialized in the production of inkjet print head, is able to deposit nucleic acids, proteins, carbohydrates, nanoparticles, blood, organic compounds.
  • the deposited minimum volume is 100 pL.
  • the inkjet technology for depositing biological material see Goldmann, T., and Gonzalez, J.S. (2000) "DNA Printing: Utilization of a Standard Inkjet Printer for the Transfer of Nucleic Acids to Solid Supports," J. Biochem. Biophys. Methods 42, pp. 105-110.
  • thermal inkjet technology cannot be used in such application, since the temperature reached by the fluid during the generation and the ejection process of the microdrop from the nozzle may both chemically and physically degrade some of the used reagents, by dissociating some of their complex molecules.
  • the device according to the present invention in addition to the print head of the aforementioned type, may also include:
  • one or more nozzles able to eject air jets in order to remove foreign substances that may be present on the slide (dust or other things).
  • the positioning system 35 is formed by a three- axis Cartesian moving system for the deposition of the reagents (see in the following, in relation to fig. 10).
  • pre-processing of the image and signal conditioning e.g. by means of pre- filtering algorithms and boundary recognition set forth above;
  • the execution of the two steps A and B is strictly correlated by means of counter-reaction mechanisms.
  • the system acquires an appropriate portion of the specimen 2D map in real time, it processes the received information and it synthesizes the control law to be sent to the reagent deposition system and to the positioning systems. Such situation is shown in fig. 6, deriving from the one showed in fig. 2.
  • Fig. 7 shows the block diagram relating to the functional architecture of the system, substantially comprising a processing unit, followed by a reagent deposition system, and a system of the source-detector type in reaction.
  • position, shape and dimensions of the specimens on the support are acquired by means of a pair source 10 - detector 11 , whose positioning with respect to the support may be either the one shown in fig. 8 (transmission functioning) or the one shown in fig. 9 (reflection functioning).
  • the source may be a solid-state laser or a LED (figures show the case of a laser) while the detector may be a photodiode or an APD (Avalanche PhotoDiode).
  • the 2D map of the specimen is obtained as the scanning of the support progresses, by moving the source/detector pair.
  • a first step of reagent deposition is performed in the already detected points according to the coloring protocol to be implemented. More in detail, according to the signal received by the detector, appropriately conditioned, which changes if the specimen is present or is absent on the support in the analyzed point, a 2D map of the position of the specimen on the support is processed in real time, and appropriate control signals are sent to the reagent deposition system.
  • Fig. 10 shows an embodiment of a device implementing the method.
  • the positioning system is formed by a Cartesian moving system whose X-axis 6 and Y-axis 7 are showed in the figure. Such system allows to correctly position the pair source-detector 10,11 and of the reagent deposition system.
  • a control system based on a computer, starting from the signal received by the detector 11 , determines exact position, geometry of the shape and dimensions of the biological specimens on the surface of the support 1 (the figure shows the case of a histological slide). According to the extracted information, the computer pilots the subsystem that deposits the reagents, formed by an inkjet print head 4 comprising an appropriate set of nozzles, connected to the different reagent tanks 3.

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  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Automatic Analysis And Handling Materials Therefor (AREA)

Abstract

Dispositif pour déposer automatiquement des réactifs sur un ou plusieurs échantillons biologiques à colorer sur une ou plusieurs lames, comprenant : des moyens d'acquisition de cartes en 2D des positions spatiales desdits échantillons biologiques à colorer sur lesdites lames; des moyens pour déposer les réactifs uniquement dans lesdites positions spatiales en fonction des informations contenues dans lesdites cartes en 2D.
PCT/IB2010/055978 2009-12-21 2010-12-21 Dispositif et procédé pour déposer automatiquement des réactifs sur des échantillons biologiques avec une précision élevée WO2011077370A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US60/288,398 2001-05-02
US28839809P 2009-12-21 2009-12-21

Publications (2)

Publication Number Publication Date
WO2011077370A2 true WO2011077370A2 (fr) 2011-06-30
WO2011077370A3 WO2011077370A3 (fr) 2011-11-24

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PCT/IB2010/055978 WO2011077370A2 (fr) 2009-12-21 2010-12-21 Dispositif et procédé pour déposer automatiquement des réactifs sur des échantillons biologiques avec une précision élevée

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5232664A (en) 1991-09-18 1993-08-03 Ventana Medical Systems, Inc. Liquid dispenser
US5595707A (en) 1990-03-02 1997-01-21 Ventana Medical Systems, Inc. Automated biological reaction apparatus

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US5839091A (en) * 1996-10-07 1998-11-17 Lab Vision Corporation Method and apparatus for automatic tissue staining
JP3902939B2 (ja) * 2001-10-26 2007-04-11 株式会社日立ハイテクノロジーズ 標本中の微小領域測定装置及び方法
DE10323776A1 (de) * 2003-05-23 2004-12-09 Vulkan Technic Maschinen-Konstruktions Gmbh Verfahren zum Reinigen von Objektträgern und Vorrichtung hierfür
JP2008275550A (ja) * 2007-05-07 2008-11-13 Canon Inc 検体の前処理方法及び検体の分析方法
DE102008018982A1 (de) * 2008-04-14 2009-11-05 Merz, Hartmut, Prof. Dr. med. Automatische Vorrichtung zur Durchführung von Nachweisreaktionen und Verfahren zur Dosierung von Reagenzien auf Objektträgern

Patent Citations (2)

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Publication number Priority date Publication date Assignee Title
US5595707A (en) 1990-03-02 1997-01-21 Ventana Medical Systems, Inc. Automated biological reaction apparatus
US5232664A (en) 1991-09-18 1993-08-03 Ventana Medical Systems, Inc. Liquid dispenser

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
GOLDMANN, T., GONZALEZ, J.S.: "DNA Printing: Utilization of a Standard Inkjet Printer for the Transfer of Nucleic Acids to Solid Supports", J. BIOCHEM. BIOPHYS. METHODS, vol. 42, 2000, pages 105 - 110, XP000889698, DOI: doi:10.1016/S0165-022X(99)00049-4

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