WO2011077370A2

WO2011077370A2 - Device and method for the automatic, high-precision and high- accuracy deposition of reagents on biological specimens

Info

Publication number: WO2011077370A2
Application number: PCT/IB2010/055978
Authority: WO
Inventors: Sergio Cometto; Pierluigi Buttu
Original assignee: Intelsint S.R.L.
Priority date: 2009-12-21
Filing date: 2010-12-21
Publication date: 2011-06-30
Also published as: WO2011077370A3

Abstract

Device for the automatic deposition of reagents on one or more biological specimens to be colored on one or more slides, comprising: 2D maps acquisition means of the spatial positions of said biological specimens to be colored on said slides, deposition means of the reagents only on said spatial positions, on the basis of the information contained in said 2D maps.

Description

DEVICE AND METHOD FOR THE AUTOMATIC, HIGH-PRECISION AND HIGH- ACCURACY DEPOSITION OF REAGENTS ON BIOLOGICAL SPECIMENS.

Application field of the invention

The present invention refers to a device and method for the automatic, high- precision and high-accuracy deposition of reagents on biological specimens.

DESCRIPTION OF THE PRIOR ART

The examination of biological specimens by means of an optical microscope is essential for diagnosing serious illnesses such as cancer and for studying the morphology of tissues and cells. 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. 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.

At present, in the field of the automatic coloring of the aforementioned biological specimens, 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.

In the past, the coloring steps of the tissues for histological or cytological analysis were performed by hand. Such procedure inevitably gave not uniform, not reproducible and hardly comparable results. Modern automatic techniques comprise two different methods to apply the reagents on the specimens. 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.

One of the biggest problems of this method is the possible cross-contamination of the specimens (namely when a portion of specimen comes off its slide and ends on the slide of another specimen) and the carry-over of the reagents (with the consequent contamination of a reagent with the previous one). A cross- contamination is possible both among the samples immersed in the same basin, and among the samples immersed in subsequent basins. In order to reduce the problem of the carry-over of the reagents, the used technique implies the simple shaking of the slide holder frame after its extraction from a basin and before its immersion in the following basin. A certain quantity of reagent, however, is carried anyway into the following basin, contaminating the reagent contained by the latter. For this reason, the reagents in the basins are regularly replaced, which results in a high waste of material.

According to a second type of instrument for the automatic coloring, 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. Thus 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, however, 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:

• pre-treatment of the sample in order to reduce non-specific bindings;

• treatment with primary antibody and incubation;

• treatment with secondary antibody bound to an enzyme and incubation;

• reaction of the substrate with the enzyme that produces a fluorophore or a chromophore showing an area of the sample having epitopes bound with the antibody;

• countercoloring.

Each one of these steps is followed by a washing step for removing the residual reagent that did not react in the previous step. Incubations are performed at high temperatures, usually around 40°, and the sample must always be protected against dehydration.

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.

In such type of machine, 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. Thus, 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.

100, 150, 200, 400 and 600 pL (DAKO);

50, 100, 200 μΙ_ (VENTANA);

Such estimations (referred to the dimension of the specimen), however, are obtained with not quantitative methods, and therefore are approximated, thus the operator has to perform a rough estimation of the quantity which most of the time results in an overabundant estimation, in order to avoid the risk of under-dosing.

Summary of the invention

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. In particular 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, according to claim 1 , 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.

The dependent claims describe the preferred embodiments of the invention, and are an integral part of this description.

Brief description of the Figures

Further purposes and advantages of the present invention will become clear from the following detailed description of a preferred embodiment (and its alternative embodiments) and the drawings that are attached hereto, which are merely illustrative and non-limitative, in which:

figures 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;

figures 8 and 9 show two embodiments of the source-detector system of fig.

7;

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;

figures 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.

In the figures the same reference numbers and letters identify the same elements or components.

Detailed description of a preferred embodiment of the invention

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.

Examples of specimen deposition on slides are showed in figures 1A, 1 B, 1C, 1 D.

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.

In the following two preferred embodiments of the present invention are described, respectively named two-time system (or separate step system) and 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).

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.

As a result, 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. In such conditions, it is possible to derive 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.

In a particular realization, 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 (phase contrast, bright field, dark field, etc.) may be used to form an image suitable to be caught by the camera.

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 (e.g. ram memory, flash memory, hard disk) are processed in order to obtain the desired characteristics (spatial position acquisition system 32) with appropriate image processing algorithms. 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, while 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, while Fig. 12B shows the same binary map obtained from the image using a segmentation algorithm by means of binarization. In the latter, 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.

Since the image acquisition was performed in known and standardized conditions, it is possible to easily calculate the scale conversion constant metric/ pixel (mm/px o im/px) to be used in order to rescale the images- themselves and to build the reference metric for determining the position of the specimens on the support, starting from a known reference system integral with the support, such as for example the one defined by the edges of the support itself.

Fig. 5 shows a block diagram of a system implementing the STEP B.

In this case 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.

In a particular embodiment, 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. In this case 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. For another example of use of 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.

The thermal inkjet technology, however, 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 for rinsing;

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).

In a particular embodiment, 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).

An embodiment of the method that is object of the invention thus comprises the following steps:

• positioning of the image acquisition system on the slide to be analyzed; · ejection of an air jet to remove dust / undesired deposits from the slide;

• image acquisition of the slide having standard position / illumination / exposition (the slide is in the middle of the image);

• pre-processing of the image and signal conditioning, e.g. by means of pre- filtering algorithms and boundary recognition set forth above;

· calculation of the scale conversion constant linear measure / pixel (e.g. mm/pixel or im/pixel) according to the image acquisition system and to the resolution being used;

• determination of the regions of support that are occupied by the specimens by means of boundary recognition / sequencing / binarization algorithms;

• determination of position and dimension of the regions occupied by the specimens, be means of a calculation of the reference metric for the specimen positioning on the slide, starting from a known reference system and integral with the support.

• Implementation of the coloring protocol to be executed, which may be of a type per se known, by piloting the reagent deposition system (inkjet print head and ancillary nozzles) on the specimens to be colored.

In an alternative embodiment, 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.

In a preferred embodiment of such configuration, position, shape and dimensions of the specimens on the support (suitable to realize the image acquisition system (31 ) and the image spatial position acquisition system (32)) 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). In such case the 2D map of the specimen is obtained as the scanning of the support progresses, by moving the source/detector pair. At the same time, 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.

The following depositions, according to the coloring protocol to be implemented, will be performed according to the information contained in the 2D map, obtained from the first scanning and stored in the memory of the processor.

Fig. 10 shows an embodiment of a device implementing the method.

In such particular embodiment, 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.

In such particular preferred embodiment of the invention, 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.

It will be apparent to the person skilled in the art that other alternative and equivalent embodiments of the invention can be conceived and reduced to practice without departing from the scope of the invention.

From the description set forth above it will be possible for the person skilled in the art to embody the invention with no need of describing further construction details.

Claims

1. 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:

- acquisition means of 2D maps of the spatial positions on said supports of said biological specimens to be colored;

- deposition means of the reagents only on said spatial positions, according to the information contained in said 2D maps.

2. Device as in claim 1 , wherein said acquisition means of 2D maps and said deposition means of the reagents comprise:

a data processing unit (34);

an image acquisition system (31 ) and a spatial position acquisition system (32) of the images with respect to said transparent supports, in order to allow the acquisition of images having standard relative position, illumination and exposition, in order to obtain a metric on the image plan to be used as a reference for calculating said spatial positions and obtain said 2D maps;

- a positioning system (35) of the image acquisition system on the supports;

- a deposition system (51 ) of the reagents only on said spatial positions.

3. Device as in claim 2, wherein said image acquisition system (31 ) comprises:

image pre-filtering means suitable to improve the signal-to-noise ratio, and image boundary recognition means;

segmentation means by means of the binarization of the image received from said pre-filtering means.

4. Device as in claim 3, wherein said image pre-filtering means are moving average filters on the pixels of the image by rows and by columns.

5. Device as in claim 1 , wherein said reagent deposition means comprise a print head of the inkjet type with piezoelectric technology.

6. Device as in claim 2, wherein said positioning system (35) comprises a three-axis Cartesian moving system.

7. Device as in claim 1 , wherein said image acquisition system (31 ) comprises a CCD or CMOS sensor.

8. Device as in claim 1 , wherein said transparent supports are slides, and said image acquisition system (31 ) and said spatial position acquisition system (32) comprise a pair source (10) - detector (11 ), positioned with respect to said slides in transmission functioning, or in reflection functioning.

9. Method for the automatic deposition of reagents on one or more biological specimens to be colored, on one or more transparent supports, using a device as in any of the previous claims, comprising the steps of:

• positioning of the image acquisition system on the supports;

• ejection of an air jet to clean the supports;

• image acquisition on the supports;

• image pre-processing and signal conditioning;

• calculation of a measurement scale conversion constant metric/pixel;

• determination of the regions of the supports occupied by the specimens;

• determination of position and dimensions of the regions occupied by the specimens on the supports;

• implementation of a specimen coloring protocol, only on said spatial positions.