Method for preparing a processed virtual analysis plate

ES 2 525 486 T3

DESCRIPTION

Preparation procedure of a treated virtual analysis plate.

The present invention relates to a method for preparing a virtual analysis plate of a cytological sample arranged on an analysis plate to allow the cellular analysis of said sample, of the type comprising the following steps:

- carry out a treatment of the sample, said treatment being carried out to allow the pathological cells to be differentiated from the healthy cells of the sample,

- carry out an acquisition of images of the sample arranged on the analysis plate in order to obtain a plurality of images that each represent an area of ​​the analysis square, said images placed one next to the other forming an image of the entire sample to create a virtual analysis plate.

Such a procedure is disclosed, for example, in document US-A-2008/0032328.

The invention is particularly applicable to cytological analysis procedures.

The analysis of the samples is used, for example, for the diagnosis of pathologies, from cells extracted by smears (cervical, vaginal or others), by puncturing organs (sinus, thyroid, lymph nodes or others) or also by collecting samples (urine, bronchoalveolar lavage or others), to detect any type of pathology and more particularly precancerous or cancerous states.

In a known manner, the samples are examined by specialized observers and prepared to detect the cells that are susceptible to being pathological in a sample placed on a test plate or slides. In order to allow the detection of potentially pathological cells, the sample undergoes a treatment, such as staining, which makes it possible to reveal, among others, the characteristics of the nucleus and cytoplasm of the cells to help localization and diagnosis. of pathological cells. When the sample is observed, the potentially pathological cells then show differences in tinctorial affinities, in size and shape, both at the level of the nucleus and the cytoplasm, with respect to normal cells.

The analysis can be carried out manually, without particular assistance. In this case, the doctor or specialized technician moves sample plates under a microscope and observes each of them to detect morphological abnormalities that indicate pathological cells that may correspond to a precancerous or cancerous state, for example. Such an analysis method is tedious and time consuming. In addition, it does not offer satisfactory results with, especially, a number of "false negatives" estimated at approximately 30%, that is, samples considered as normal, although there is a pathology in the patient, particularly precancerous or cancerous, with the risks of evolution of cancer in a wrongly reassured patient.

To improve the results of the analysis, it has been proposed to improve the sampling, that is, the number of cells, their fixation, their coloration and their spread on the analysis plate, but also to help the doctor or specialized technician in their analysis, for For example, thanks to computerized means of analysis, such as image processing programs and others.

For this, a camera or an image-taking apparatus is used to acquire images of the different areas of the sample arranged on the analysis plate and communicate the data of these images to a computer system that then works on an analysis plate. "virtual".

This computer system allows a signal processing, a pre-treatment of the images and a comparative analysis of the images with, eventually, newly created or existing databases to speed up the analysis process and thus allow the analysis of a higher number. samples and to assist the doctor or specialized technician. The images of a sample are examined, for example, automatically and if some areas that present an abnormality are located, the corresponding images are communicated to a doctor or a specialized technician who can then determine whether or not these areas show pathological cells. The doctor or specialized technician then observes only some abnormal areas without analyzing the areas considered normal by the computer system. Such a procedure effectively makes it possible to speed up the analysis and to guarantee the reliability of the diagnosis.

However, the doctor or specialized technician then no longer has the opportunity to observe normal samples or those showing minor morphological modifications, which is detrimental to their appreciation of the samples and especially to their learning curve, including conservation. of your diagnostic acuity. In effect, sample analysis is based on the training and practice of the specialized physician or technician to examine samples and compare normal areas and areas showing abnormalities. The fact

ES 2 525 486 T3 of suppressing this practice by computer-aided analysis can lead to doctors or specialized technicians to lose their skills and thus lead to analysis errors.

In addition, the line-by-line and not field-by-field scanning operation for a color image requires an overwriting or a red / green / blue (RVB) recalibration which requires a high level of adjustment for the recalibration of the images, this being high adjustment level sensitive to the slightest vibrations. Thus, this line-by-line scanning for a color image can be time consuming, due to the treatments applied to the slide during scanning. Furthermore, the scanning operation for a color image requires significant image compression times.

On the other hand, the digitization operation is delicate for samples that contain three-dimensional groupings of cells, that is to say stacks of cells, particularly pathological cells. In effect, the camera's focus is performed at a cluster thickness defined by one or more focus points at the cluster level, leaving the other deeper or shallower points out of focus. In order to obtain a wide image field, that is to say of the entire analysis plate, suitable for a detection or diagnostic reading on the single virtual plate, it is necessary to have as small an out-of-focus surface as possible (less than 5%). In addition, segmentation tools are more difficult to handle on blurred areas, affecting both single cells and three-dimensional clusters. In this way, the analysis of cells and nuclei becomes much less relevant and can even lead to poor categorization of cells.

The invention aims to alleviate the aforementioned drawbacks, proposing an analysis procedure that, while saving time in digitization processes, makes them more efficient in terms of the analyzable surface and more efficient in terms of the quality of detection of cellular anomalies. for computer-aided analysis, in a more relevant quality monitoring environment that associates diagnostic verification and education points with diagnosis for the doctor or specialized technician.

To this end, the invention refers to a process of the aforementioned type, characterized in that it comprises the following steps:

- locating a reference plane of the analysis plate comprising a slide and a coverslip arranged above the slide, for the acquisition of images, said reference plane being defined by the surface of the slide or coverslip; Y

- carry out at least one second image acquisition, said second acquisition being carried out at a different thickness from the sample of the first acquisition, to obtain a plurality of images, corresponding to a section of the sample made with a different thickness.

Such a method makes it possible to make at least one image of a sample in a particularly simple manner, making it possible to detect possibly pathological cells and to acquire a large number of information about the sample, as will be described later.

According to other characteristics of the preparation procedure:

- the location of the reference plane of the analysis plate is carried out by acquiring a topographic image of the slide or coverslip,

- the analysis plate comprises at least one mark arranged to locate the reference plane for the acquisition of images regardless of the thickness of the sample arranged on the analysis plate, said image acquisition being carried out at a defined thickness with respect to to the reference plane of the slide or coverslip defined by the mark,

- the analysis plate comprises at least four marks arranged around the sample,

- the or each mark is printed on the analysis plate,

- the thickness of the imaging sample is adjustable,

- the method also comprises a step of superimposing the data of the images acquired during the acquisition of the images and those of the acquired images modified during the processing of the acquired images, to produce a single wide-field virtual image plane,

- the acquisition of images of the sample placed on the analysis plate is carried out according to a scale of gray levels,

- the procedure also includes the following step:

ES 2 525 486 T3

- carry out on the virtual analysis plate (2) a treatment of the acquired images to obtain a virtual restitution of the colors and the intensity of the colors of the cytoplasm and / or the nucleus of the cells of the sample, said colors and said intensity be modified according to the preferences of the person in charge of the analysis,

- the treatment of the sample is a nuclear staining stage or a cytology staining, said treatment being arranged to make the cytoplasm almost transparent and accentuate the contrast between the nucleus and / or the cytoplasmic RNA of the cells and the cytoplasm,

- the treatment of the acquired images corresponds to a virtual polychrome recoloring of the Papanicolaou, Schorr, MAy Grunwald Giemsa or Giemsa type or monochrome of these acquired images,

- the level of virtual recoloring of the acquired images is adjustable,

- the procedure comprises a stage of visualization of the virtual analysis plate,

- the procedure comprises the following steps:

- automatically scroll the acquired wide-field images of the sample, without displaying complementary data; Y

- automatically stop the parade if at least one anomaly is detected by the automatic analysis system that could mean the presence of a pathological cell,

- the automatic interruption of the parade, if at least one anomaly is detected that may mean the presence of a pathological cell, comprises a stage of displaying the analysis plate enlarged at the level of the anomaly and secondary display of information about the area displayed sample, and / or on the entire sample, and / or on the patient on whom the sample has been made, and / or on the result of complementary examinations carried out on the sample,

- the method comprises a stage of enlarging the virtual analysis plate in order to allow the visualization of a detail of said plate.

Other aspects and advantages of the invention will appear on reading the following description, given by way of example and made with reference to the drawings, in which:

figure 1 is a schematic representation of the different stages of a procedure for preparing a virtual analysis plate according to the invention,

FIG. 2 is a schematic sectional representation of a sample analysis plate and a slide digitizing device, and

figure 3 is a diagram representing the different stages of analysis of a virtual plate.

With reference to FIG. 1, a procedure for preparing a virtual analysis plate 2 for its cellular analysis assisted by a computer system is described. By virtual plate 2, it is understood a set of information and grouped numerical data that refer to a sample 4.

Sample 4 is obtained, for example, by smears (cervical, vaginal or others), by organ puncture (sinus, thyroid, ganglion or others) or also by collecting samples (urine, bronchoalveolar lavage or others). During a first stage A, the sample is suspended, for example in a sample tube or bottle 6.

During a stage B, the sample 4 is placed on an analysis plate 8. In a known manner, the deposition of the cells on the plate 8 is carried out, for example, by decantation. The sample is poured into a settling chamber 10 the bottom of which opens onto the test plate 8. Absorption means 12 allow the solution to be absorbed as the cells settle on the test plate 8. Such a deposition procedure is known and will not be described in detail here.

During a stage C, the sample undergoes a treatment that aims to mark / stain the nuclei, DNA and / or RNA of the cells, accentuating very clearly the contrast with respect to the cytoplasms of these same cells. Such marking makes it possible to correctly segment the nuclei to carry out the morphological study and to quantify the DNA (for a ploidy analysis, for example) and / or the RNA to locate potentially pathological cells.

This marking is carried out, for example, automatically by means of an automaton equipped with pipetting means used at the same time to put the sample 4 in solution and to deposit the cell suspension on a

ES 2 525 486 T3 analysis plate 8.

Stage B of decantation of the cell suspension can be carried out before or after stage C of marking / staining described above.

This marking is preferably carried out with hematoxylin.

A marker other than the markers used for DNA quantification can be used, such as hypericin or a specific marker for proliferation or pathogens, such as, for example, oncogenic viruses of the Human Papilloma Virus type, in the case of the smear of the cervix.

Other colorations used in the state of the art could be used, but they have drawbacks. Thus, a stoichiometric staining could be considered, which offers a staining of the nuclei proportional to the amount of DNA, allowing their quantification, and therefore locating and analyzing pathological cells in the field of ploidy.

However, this particular staining, when it comes to a Feulgen stain for example, is technically incompatible with a Papanicolaou stain and therefore needs to repeat a cell diffusion on the slide for analysis by the doctor or specialized technician. Certain industrialists have tried to associate a stoichiometric staining with the Papanicolaou stain and have therefore used a stain that contains a thionin and that requires fixation with methanol, which is toxic, and above all that modifies the Papanicolaou staining in its interpretation, in particularly with nuclei whose chromatin appears too black for a fine analysis of the composition of said nuclei, and therefore entails a difficulty of analysis for the diagnosis of pre-cancerous or cancerous states.

During a stage D, the analysis plate 8 comprising sample 4, stained by a known nuclear stain or by a known cytological stain but modified to make the cytoplasm almost transparent, is subjected to illumination with white light 14 to acquire images of sample 4 by means of image acquisition device 16.

The acquisition of images in white light makes it possible to obtain images of the sample stained by nuclear staining or modified cytological staining, preserving almost transparent cytoplasms to increase the contrast with the nuclei.

The image acquisition apparatus 16 makes it possible to obtain images in black and white, more precisely on a scale of gray levels. Such selection allows faster image acquisition and better resolution than if the acquisition is done in color.

The acquisition of images in white light requires having previously located a reference plane of the analysis plate, which defines a reference thickness with respect to which the thickness of the sample is measured, by way of typographic tracing of the surface and of their inequalities.

Referring to Figure 2, the analysis plate 8 comprises a slide 20 and a coverslip 22 arranged on top of the slide 20. The sample 4 from the extraction is disposed between the slide 20 and the coverslip 22, and is held by the adhesive 24 .

The reference plane of the analysis plate 8 is defined either at the level of the upper face of the slide 20, designated by the thickness Za, or at the level of the upper face of the slide 22 designated by the thickness Zb.

According to one embodiment, the location of the reference plane is obtained by acquiring an image of the slide or the coverslip, for example by ultrasound or light reflection on the slide or the coverslip with or without a specific treatment of the slide.

This treatment consists for example in making the slide or coverslip at least partially reflective.

Before the acquisition of images in white light, the location of the reference plane of the analysis plate allows to avoid the focus on the cells and the problems of polymerization of the adhesives for the glass slides, or any bubbles present in the sample . The acquisition of images in white light allows to obtain, in different localized thicknesses with respect to the reference plane, several image planes for a secondarily treated focus, to make it. It also makes it possible to get rid of the problems of horizontal adjustment of the device. Indeed, the definition of the reference plane makes it possible to recalibrate the image with respect to this plane.

Thus, the adjustment of the apparatus with respect to the plate does not need as great a precision as in the prior art.

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According to another embodiment, the analysis plate 8 comprises at least one mark 13 arranged to locate the reference plane of the analysis plate before the acquisition of images of the sample.

According to a variant, the analysis plate 8 comprises at least four marks 13 arranged evenly distributed around the sample, for example the four cardinal points. According to one embodiment, the or each marking 13 is printed on the analysis plate 8. According to another embodiment, the or each marking 13 is fixed on the analysis plate 8, on the slide or the coverslip.

Furthermore, in order to correctly visualize clusters of three-dimensional cells during digitization, image acquisition is performed at one or more different thicknesses of the sample.

These different thicknesses at which image acquisition is performed can be selected prior to acquisition and are located with respect to the reference plane on the analysis slide defined by the mark or by the image of the slide or coverslip surface.

The image acquisition apparatus 16 makes it possible to scan the sample 4 with a very fine resolution and to obtain images in white light from a plate 8. The analysis board 8 is scanned line by line as proposed by many industrialists. Thus, each acquired image represents a band 18 of predefined width of the analysis plate 8, for example a width of low value is selected in order to obtain a large number of images and therefore a better resolution. The images placed side by side allow to obtain an image of the entire sample plate, or wide-field images, and therefore of the entire sample in order to form a virtual analysis plate 2, as represented in step E of figure 1. Where appropriate, the acquisition allows images of the entire sample plate to be obtained at different thickness of the sample.

Thus, the images, obtained with one and the same apparatus and in a very simple way, are a faithful representation of sample 4, which associates a large number of information in white light.

The numerical data obtained with the apparatus 16 in white light undergoes a computer treatment to obtain a modified virtual analysis plate 2 formed from the image (s) that have undergone a virtual polychrome recoloring of the Papanicolaou, Schorr, May Grunwald Giemsa or Giemsa type or monochrome, which is intended to stain the cells as described and conventionally known for cytological or morphological analysis by the person skilled in the art.

According to a computerized treatment method, virtual recoloring is a Papanicolaou stain, obtained as it has been known for a long time and whose semiology, widely described in the bibliography, allows the eventual recognition of cytoplasmic and nuclear abnormalities corresponding for example to the presence of pre-cancerous or cancerous cells. This recoloration also allows the recognition of the different types of cells and their number, to determine the representative quality of the sample and, for example, to define whether the sample is representative or not. This virtual recoloring allows analysis of the virtual plaque using a cell analysis procedure described below. Papanicolaou recoloring being virtual, it can be considered a Feulgen type coloration without any compatibility problem.

Cellular analysis is carried out by examining the images of the sample by the doctor or specialized technician in charge of detecting pathological cells, to propose a diagnosis that will eventually initiate deeper examinations, including treatment. For quality assurance reasons, the presence of a doctor or a specialized technician is mandatory so that the detection of possibly pathological cells cannot be fully automated.

With reference to figure 3, the visualization and diagnosis process of the images that have undergone recoloration is detailed.

During a stage F, the images 30 of the modified virtual analysis plate 2 formed with the image, or of the images in different thicknesses of the sample, which have undergone virtual recoloring, are projected onto a display medium such as a screen. . The virtual restitution of colors and their intensity, both for the cytoplasm and for the nucleus, can be adjusted so that it corresponds as closely as possible to the habits of each reader, that is, the doctor or the specialized technician, in terms of intensity and quality of coloring.

The information 31 on the patient can be associated to the virtual analysis plate 2 by entering this information in a database and associating it with the image, or images of different thicknesses, of the sample 4 corresponding to the patient on which it has been This sample 4 was carried out after authorization from the operator, for example by pressing a virtual button 32 during step F.

The images 34 of the sample are moved under the eyes of the doctor or the specialized technician for examination during a stage G. The parade of the images 34 is organized by the computer system and is carried out in

ES 2 525 486 T3 automatic way. Each image 34 is displayed completely, without visualization of additional data about the patient, in order to avoid the dispersion of the attention of the doctor or the specialized technician during the analysis (or “screening” in English), and this during a predetermined time calculated to allow the doctor or the specialized technician to observe the totality of each projected image and detect a possible anomaly in an image. The display time of each image can be adjusted by the doctor or the specialized technician according to their skills or according to other information. For example, if the sample was taken for a "risk" patient who has a higher probability of presenting pathological cells, the display time can be adjusted to be longer, in order to carry out a more detailed examination. of the sample.

The images 34 that move are therefore those of the modified virtual analysis plate, formed from the image or images in different thicknesses, which have undergone virtual recoloring of the Papanicolaou, Schorr, May Gruwald Giemsa or Giemsa type. The images 34 under this staining make it possible to detect the eventual presence of anomalies corresponding, for example, to the presence of precancerous or cancerous cells and whose semiology, known for a long time, is widely described in the bibliography, and to verify that the sample responds really up to the Bethesda criteria, in the area of ​​cervical smear for example. The program can also acquire other information on cell broadcasting. This information is related to the image (or images of different thicknesses) by the computer system to complete the virtual analysis plate 2.

The doctor or specialized technician observes each image displayed, on the screen, and determines whether an abnormality is present or not. This detection can also be carried out automatically by the computer system through the image analysis program.

In the case in which neither the doctor or the specialized technician, nor the cellular analysis computer system detects an abnormality in the displayed image 38 during an H stage, the diagnostic procedure is continued by projecting the next image, after a previously set display time elapses.

According to one mode of carrying out the cellular analysis, a stage is provided during which the computer system automatically stops the parade of images on an image on which no anomaly is detected, and awaits a validation from the doctor or specialized technician to return to start the parade. By stopping on such an image, it is possible to analyze in detail some so-called reference images, in order to confirm what is considered a normal sample. The stop on so-called normal images can be performed randomly or at the end of a certain number of displayed images. The description of the detailed analysis of an image follows in connection with the analysis of an image that exhibits an anomaly.

If an anomaly is detected in the image 40 by the doctor or the specialized technician and / or the computer system, the procedure continues by interrupting the parade of the images 36 during a stage I.

The parade is interrupted automatically by the computer system if it detects an anomaly, or manually by the doctor or specialized technician if you wish to observe a more detailed image or if an anomaly is located.

Image 42 is then displayed at a higher magnification percentage, in order to visualize the anomaly in detail. For example, the visualization of the images can be done with a magnification percentage x10 during the movement, and be doubled (x20) in the case in which an anomaly is detected, as represented in step J of figure 3.

The display of the image containing an anomaly may be accompanied by the display of information 44 on the area of ​​the displayed sample and / or on the entire sample and / or on the patient from whom the sample is taken and / or on the result of complementary tests carried out on the sample, in particular molecular biology tests.

The image shown under virtual recoloration, and for which the parade has been interrupted, is associated with the image of the same area without virtual recoloration. This visualization allows the doctor or the specialized technician to refine his analysis of the visualized cells and to confirm or not if some are eventually pathological. Furthermore, with the visualization of the image without virtual recoloring, other information can be simultaneously displayed, such as quantitative data, spectra or information about the patient from whom the sample was taken, etc. This finer analysis, coupled with the automated stop of the parade, reduces the number of false negatives. Furthermore, in the field of cervical smears, for example, the control of the diagnosis by the doctor or the specialized technician of the areas selected by the system makes it possible to preserve a high level of specificity of the cytological diagnosis in this case. Thus, the sensitivity and specificity criteria of the detection smear become closer and higher.

The restart of the parade can only be controlled by the doctor or the specialized technician, for example by pressing a validation button 46. This makes it possible to make sure that if the parade has been interrupted automatically, the image displayed and eventually presented an abnormality has been examined either by a doctor or

ES 2 525 486 T3 a specialized technician. It is the same for the normal images displayed.

During stages F to J, the doctor or specialized technician can freely make an enlargement of particular areas of the displayed image, both in the image with virtual recolorization and in the image without virtual recoloration. The physician or specialist technician can also change from the virtual recolorized image to the non-virtual recolorized image as desired.

The procedure described above allows fast and efficient analysis of samples, reducing the risk of false negatives.

In addition, the experience of specialized doctors or technicians in terms of diagnostic quality and, above all, specificity, is preserved due to the recognition of colors and their intensity in relation to the virtual images of the cytological and / or histological preparations treated and visualized.

The procedure also allows the analysis plate to be adapted to the preferences of the physician or specialized technician in charge of the analysis. Indeed, the doctor or specialized technician can select the intensity and color of the virtual analysis plate at will.

The procedure also makes it possible to acquire images of better resolution in gray levels than through color acquisition.

In addition, the procedure allows an acquisition in different thicknesses in the samples, while preserving clear, correct images, thanks to the reference plane that allows to get rid of the problem of horizontal adjustment of the apparatus. Thus, even if the apparatus does not allow a sufficiently horizontal slide of the slide due to lack of adjustments, it is possible to obtain an image of interpretable quality. As the adjustment of the apparatus does not require such high precision, the procedure offers a first time saving for digitization.

Acquisition in gray levels also allows faster acquisition than in color, particularly during acquisition at different thicknesses in the sample. Indeed, when the doctor or the specialized technician in charge of the analysis wishes to make, for example, a few planes every 2 pm in the thickness of a cell, that is to say 10 pm, the acquisition is five times longer, which is too long to a single sample. At gray levels, the acquisition time is substantially reduced.

Finally, this procedure, which allows recoloring of a virtual analysis plate, allows saving dye while guaranteeing fast and efficient analysis of the samples.