WO2013107463A1

WO2013107463A1 - System and method for standardized, optimized and pre-configured quantitative digital pathology

Info

Publication number: WO2013107463A1
Application number: PCT/DK2013/050018
Authority: WO
Inventors: Michael Grunkin; Johan Doré HANSEN
Original assignee: Visiopharm A/S
Priority date: 2012-01-20
Filing date: 2013-01-21
Publication date: 2013-07-25

Abstract

The invention is useful in quantitative digital pathology and provides a standardized method for preparing biological samples, acquiring digital images, and analysing the images through the use of a system for standardized, optimized and pre-configured quantitative digital pathology on biological samples. The system comprises a library of application packages wherein each application package sample preparation protocols for preparation of a biological sample, and at least one digital slide showing a biological sample prepared according to the instructions, instructions for image acquisition using an optimized image analysis algorithm optimized and documented based on data being at least one biological sample prepared according to the instructions and imaged according to the instructions. The application package also comprises at least one corresponding reference standard of the biological sample, an application description comprising at least information of the preparation protocol and the instructions for image acquisition. Furthermore, the system comprises a webbased presentation of the application description of a plurality of application packages in the library, said webbased presentation comprising an access request capability. The method may be executed on any suitable platform, and is particularly relevant for executing as a cloud application.

Description

System and method for standardized, optimized and pre-configured quantitative digital pathology The present invention relates to a system and a method for standardized, optimized and pre- configured digital pathology.

Background Examination of various biological tissues often includes histological examination of a tissue or cell sample removed from a patient. Such histological examination entails tissue-staining procedures that allow the morphological features of the tissue to be readily examined under a microscope. There are real challenges and problems associated with the correct quantification of structural properties of tissues, of which some are related to expensive and labor intensive procedures. Human operators are required for accessing the physical slide and mounting it on a stage under the microscope. Significant time is used for focusing and other adjustments before counting can commence. Then the pathologist, after having examined the stained tissue or cell sample, may make qualitative or quantitative determinations of the state of the tissue or the patient from whom the sample was removed.

The ability to provide quantitative statements about micro-structural tissue properties is becoming increasingly important in biopharmaceutical research and development in diverse applications related to both safety and efficacy pharmacology. Typically it is of interest to make quantitative statements about number, length, surface area, and volume of structural features reflecting the condition of an organ. It may even be of interest to investigate second-order properties based on the (number- or volume weighted) distribution of structural features. Digital pathology

In order to reduce the labor intensive procedures, visual examination of tissue and cell samples is increasingly being augmented by the use of an automated (computer-aided) image analysis system, also called digital pathology. A representative system includes a computer that receives a magnified digital representation of the tissue or cell sample from a camera and processes the received digital representation. Virtual or digital slide systems utilize, often automated, digital slide scanners that create a digital image file of an entire glass slide (whole slide image).

This is also called virtual microscopy because the images are viewed without the use of a microscope or slides. The digital slides (images) are normally maintained in an information management system that allows for archival and intelligent retrieval. The digital image files that can be visualized and navigated, just as Google Earth, allowing pathologists to use computers for warping from a low-magnitude overview image of an entire tissue specimen, into a high- magnitude field-of-view, where minute cell structures can be scrutinized.

The ultimate vision of Digital Pathology is to provide a seamless workflow from tissue preparation to histo-morphometric end-points. However, at present there are no standards for tissue preparation resulting in tremendous variability in the visual appearance of stained tissue sections leading to problems in the downstream analysis of the images.

Summary of the invention

The present invention provides a solution to the lack of a standardized method for quantitative digital pathology including a standardized method for preparing the biological sample, acquiring the digital image, and analysing the image. Accordingly, the invention relates in one aspect to a system for standardized, optimized and pre-configured quantitative digital pathology on biological samples comprising a library of application packages wherein each application package comprises

a) a verifiable and optimized biological sample preparation protocol comprising

1) instructions for preparation of a biological sample

at least one digital slide showing a biological sample prepared according to the instructions

b) instructions for image acquisition,

c) an optimized image analysis algorithm comprising image analysis instructions, wherein the algorithm is optimized and documented based on data being:

1) at least one biological sample prepared according to the instructions as defined in a) and imaged according to the instructions as defined in b) at least one corresponding reference standard of the at least one biological sample in c)1)

d) an application description comprising at least

1) information of the preparation protocol as defined in a) and the

instructions for image acquisition as defined in b) a webbased presentation of the application description as defined in d) of a plurality of application packages in the library, said webbased presentation comprising an access request capability, an execution platform providing capabilities of a) viewing digital slides

b) viewing instructions as defined in a), b) and c)

c) executing the algorithm, whereby the accessibility of the part of the application package as defined in a)-c) requires request of access through the access request capability.

Furthermore, in another aspect the invention relates to a method for a user for viewing a digital slide representing structural content of a biological sample, in a system as defined above searching the webbased presentation of application descriptions and selecting an application description corresponding to a specific structural content, requesting access through the access request capability, viewing the digital slide(s) corresponding to the selected application description. Figures

Figure 1 shows a webbased presentation of a plurality of application description fronts showing different specific applications. Figure 2 shows an example of a staining protocol as part of a sample preparation protocol

Figure 3 shows a schematic view of the system according to the invention including

a) a verifiable and optimized biological sample preparation protocol (tissue

preparation protocol)

b) instructions for image acquisition (Optimized image instructions)

c) an optimized image analysis algorithm comprising image analysis instructions d) an application description (web presentation application descriptions)

In addition the system shown includes Clinical Documentation meant to be shown to the user to support the validity of the instructions and algorithm(s) Description of the invention

Quantitative digital pathology

As discussed above there has been a total lack of standards in staining of biological samples resulting in variability in the visual appearance of stained samples and consequently problems when interpreting digital images (digital slides) of the samples. For example if the visual appearance of cancer cells in a tissue varies from slide to slide due to variability in the staining procedures then the image analysis algorithm may not be able to discriminate between cancer cells and non-cancer cells. Consequently successful quantification relies heavily on the initial steps in the workflow of digital pathology.

The present invention provides a solution to the provision of standardized quantitative digital pathology by providing means for standardizing the whole workflow from tissue preparation to analysis of the digital slide and eventually quantification. In the present context the term standardized is used to describe that the image analysis may be standardized due to the fact that the digital slides are obtained by following the instructions for preparing the biological sample and for acquiring the digital slide of the biological sample.

The solution is a system comprising a library of application packages wherein each application package comprises

a) a verifiable and optimized biological sample preparation protocol

b) instructions for image acquisition,

c) an optimized image analysis algorithm comprising image analysis instructions, d) an application description a webbased presentation of the application description as defined in d) of a plurality of application packages in the library comprising access request capability an execution platform

A schematic view of the system is shown in Figure 3.

By using the system the user is provided with all instructions for preparing the sample as well as the digital slide followed by automated image analysis which is optimised towards the instruction for preparation and consequently it is possible to standardize all the variables during preparations whereby the automated image analysis may be carried out on digital slides not suffering from a great variability. Accordingly, in the present context the term "optimized image analysis" means that the algorithm is optimized towards slides prepared according to the instructions leading to an optimal image analysis of slides prepared according to the instructions. Thus, the present invention provides a system and a method standardizing and optimizing all aspects of tissue collection, fixation, section thickness, morphologic criteria for assessment, staining processes, digitization of images, and image analysis.

The quantitative digital pathology may relate to image analysis in general, and includes in one embodiment stereology. Stereology may be defined as "the spatial interpretation of sections". It is concerned with the three-dimensional interpretation of planar sections of tissues. It provides practical techniques for extracting quantitative information about a three-dimensional material from measurements made on two-dimensional planar sections of the material (see examples below). Stereology is a method that utilizes random, systematic sampling to provide unbiased and quantitative data.

Furthermore, the system is pre-configured which in the present context means that it comprises all the steps needed for execution of the algorithms.

Biological sample

The biological sample according to the invention may be any biological sample for which there is an interest for quantification. Normally, the biological sample is a tissue sample or a body fluid sample, or a cell sample. The sample is mostly stained with at least one chemical marker, such as at least one fluorescent marker. It is preferred that the sample is illuminated while the images are obtained, such as by using UV or illumination visible to the human eye. Tissue samples are normally prepared as tissue section, such as a histological section. Library of application packages

The standardization is obtained by the provision of a library of application packages, wherein each package provides instructions as well as algorithms necessary to carry out the quantitative digital pathology for one specific biological sample, and there is a one-to-one correlation between each of the items in an application package.

Examples of various application packages are shown in Figure 1 .

Application package

As described above each application package comprises a) a verifiable and optimized biological sample preparation protocol

b) instructions for image acquisition,

c) an optimized image analysis algorithm comprising image analysis instructions, d) an application description

The image analysis algorithm is optimized towards the sample preparation protocol, and both the algorithm and the sample preparation protocol are optimized through a scientific process and verified on a dataset from clinical testing wherein the dataset is sufficient for providing the relevant statistical results.

Biological sample preparation protocol

The instructions for preparation of a biological sample includes the relevant steps for securing a standardized and optimized preparation, including but not limited to tissue collection, fixation, section thickness, and the staining processes itself. Preferably, the preparation protocol comprises instructions for preparation methods selected from the group consisting of sample collection, sample trimming, sample fixation, sample embedding, sample sectioning, sample staining and sample storage.

Each protocol is optimized to the specific biological sample, such as the specific tissue and the specific types of cells or other structures to be enhanced and quantified. Examples of protocols are for example found here: vww. nor jQC.org and one example of a protocol is shown in Figure 2.

Furthermore, the protocol also includes an interactive aspect in that the protocol provides the customer with at least one relevant digital slide showing a sample of tissue optimally prepared according to the instructions. These slides will allow the user to compare with slides produced internally in their lab, and thereby validate whether the staining procedure has been followed correctly or whether their own slides have been stained too much or too little.

This aspect of the optimized protocols will eliminate a significant part of the variability at the front-end of the process leading to a much higher precision in the quantification, and make end- points based upon morphometry and staining intensity far more useful.

Image acquisition

In addition to the standardized and optimized preparation of the biological samples it is also important in order to obtain optimized digital slides that the image acquisition is standardized and optimized as well. Preferably, the instructions for image acquisition comprises instructions for acquisition selected from the group consisting of magnification, exposure time, modality, resolution, gamma, gain, calibration, scanner, and slides or combinations thereof. In addition to the image analysis algorithm the application package may contain one or more auxiliary algorithms, such as an auxiliary algorithm for region-of-interest (ROI) detection, and/or removal of artefacts.

Furthermore, for specific users the algorithms may be tuned or customized to specific user variables.

Image analysis

The application package furthermore comprises an optimized image analysis algorithm. The algorithm is optimized and documented based on data being: at least one biological sample prepared according to the instructions in the sample preparation protocol and imaged according to the instructions for image acquisition, - at least one corresponding reference standard of the at least one biological sample

By first ensuring that the biological sample is prepared according to a protocol which is optimized towards the specific sample and the image acquisition is standardized according to the instruction, and then using an image analysis algorithm which is optimized towards a biological sample which is prepared and imaged according to the standardized instructions then the problems of variability as discussed above have been solved or at least diminished to a level acceptable for obtaining correct results during the image analysis.

Furthermore, the intended purpose of the image analysis algorithm is to provide end-points that are concordant with pathologists or preferably a "Gold-standard", if such exist, referred to as the reference standard. Accordingly, the image analysis algorithm is also optimized and documented based on at least one corresponding reference standard of the at least one biological sample which means that the results obtained using the image analysis algorithm are concordant with results obtained by for example a pathologist on the same slides.

Both the optimization towards the preparation protocol and the reference standard is preferably documented in the application package as well. Typically the algorithm is developed on one development dataset and verified on another verification dataset using the reference standards. The relevant reference standards may be outlining of relevant tissue Regions Of Interest (e.g. tumor regions) linked with a grade, score, rank, number/count, area, circumference, length, and/or thickness, or other relevant end-point. Preferably the reference standard is established by a human, such as wherein reference standard is established by a human as a grade, score, rank, number/count, area,

circumference, length, and/or thickness.

Application description The system provides a library targeting highly specialized research applications each being described in a unique application description and the library may be searched based on the application descriptions, wherein each application description corresponds to a specific structural content of a specific biological sample. The application description contains information about the capabilities of the application package, such as highly specialized information of how it is analyzing the image, the computed end-points, screenshots, links to scientific literature, Webinars describing the application, user manual, information about with whom the application was developed, and a listing of other users.

Webbased presentation The user accesses the library through the Internet where a plurality of application descriptions is accessible freely. Accordingly, the system further comprises a webbased presentation of a plurality of application descriptions as well as an access request capability. Thereby the user may browse through the various application descriptions in search of an application description suitable for his or her purpose. Once a suitable application description is identified then the user may request access through the access request capability. In one embodiment the access request capability is a button or a link to be activated and which allows the user to access the rest of the application package, since the accessibility of the part of the application package comprising the sample preparation protocol, the instructions for image acquisition as well as the image analysis algorithm requires request of access through the access request capability. The access may be granted for a period of time, or for a number of slides analysed or any other limitation that is suitable.

Execution platform For viewing and executing the system also comprises an execution platform providing capabilities of a) viewing digital slides b) viewing instructions for as defined above, and

c) executing the algorithm.

Preferably also, the execution platform provides capabilities for viewing the results from executing the algorithm, ie. the image analysis results.

The execution platform may be provided at any suitable position, such as at the user end as a deployed application, ie. a client application. When the user requests and is granted access to the part of the application package not readily available from the webbased application in such scenario then the algorithm is executed through the application at the user end.

It is preferred to provide the software related to the system as a Software-as-a-Service (SaaS), ie, "on-demand software" being a software delivery model in which software and its associated data are hosted centrally, typically in a cloud. Accordingly, in a preferred embodiment the execution platform is provided as a cloud application service or also denoted cloud based software delivery. Cloud computing may be defined as a model for enabling convenient, on- demand network access to a shared pool of configurable computing resources (e.g., networks, servers, storage, applications, and services). Independent of location the execution platform provides capabilities of viewing and analysing the user's digital slides. In a preferred embodiment the execution platform also provides capabilities of viewing the at least one digital slide provided with the sample preparation protocol. The digital slides may be located at user end or uploaded to a server hosting the execution platform. In a preferred embodiment wherein the execution platform is provided as a cloud application service, the slides may be uploaded to the cloud or the execution platform may further provide capabilities of viewing digital slides positioned on a remote computer system. Although the image analysis, optionally including stereology, may be provided as a Web application, it is preferred to base the software delivery on Application Virtualization rather than on Web applications. Application virtualization is defined as software technologies that improve portability, manageability and compatibility of applications by encapsulating them from the underlying operating system on which they are executed. A fully virtualized application is not installed in the traditional sense, although it is still executed as if it were. The application is fooled at runtime into believing that it is directly interfacing with the original operating system and all the resources managed by it, when in reality it is not. For example the recently released Citrix XenApp technology (2010) may be used for this purpose. There are a number of technical advantages compared to Web Applications: 1 . Applications are not limited by the web browser technology. Virtualized Applications is developed as normal client application and not as web applications. This allows developers to utilize a much wider range of development tools and technologies. 2. Virtualized applications increases focus on application features and functionality. Virtualized applications have no application specific client/server communication, and the significant development resources used client/server communication can be used on features and functionalities directly benefitting users.

3. Virtualized applications eliminate web browser compatibility issues. Virtualized application is does not rely on the web browser so the application will run no matter which web browser has been installed, e.g. Internet explorer 7, 8, 9... , Mozolla, Crome etc.

4. Virtualized applications minimizes client side IT installation and maintenance issues. Virtualized application does not require installation of Java, Flash... or other common dependencies on the client computer.

Method for viewing a digital slide

The invention further provides a method for a user for viewing a digital slide representing structural content of a biological sample, said method comprising in a system as defined above searching the webbased presentation of application descriptions and selecting an application description corresponding to a specific structural content, requesting access through the access request capability, viewing the digital slide(s) corresponding to the selected application description. The user may start accessing the system for testing the slides available in the sample preparation protocol and comparing with his or her own slides. However, the main purpose of the system is for use in the analysis of the user's slides prepared according to the standardized and optimized sample preparation protocol and obtained through an image acquisition process according to the image acquisition instructions whereby the analysis is carried out using the optimized image analysis algorithm. At any time the user may compare the digital slides prepared at their end with the digital slides in the sample preparation protocol to ensure that the quality of their digital slides is acceptable. In a preferred embodiment the method further includes quantitative digital pathology on the biological sample. In one embodiment the quantitative digital pathology includes stereology. Accordingly, in one embodiment the method includes preparation of at least one slide at the user end, wherein the slide is prepared according to the standardized and optimized sample preparation protocol.

In a further embodiment the method includes acquisition of images of the slides, wherein the acquisition is performed in accordance with the image acquisition instructions.

In yet a further embodiment the method includes image analysis of the images of the slides prepared according to the standardized and optimized sample preparation protocol, wherein the image analysis is performed using the optimized image analysis algorithm.

A system and a method for acquiring digital slides as well as image analysis is described in for example PCT/DK2007/050171 , which is hereby incorporated by reference.

As discussed above the execution platform may be provided as a cloud application service, or as a deployed application.

It is preferred that the execution platform further provides capabilities of viewing digital slides positioned on a remote computer system, such as wherein the digital slides are uploaded from the user to the cloud.

The sample may be any of the samples discussed above.

Claims

1 . A system for standardized, optimized and pre-configured quantitative digital pathology on biological samples comprising a library of application packages wherein each application package comprises

a) a verifiable and optimized biological sample preparation protocol comprising

1) instructions for preparation of a biological sample

2) at least one digital slide showing a biological sample prepared

according to the instructions

b) instructions for image acquisition,

1) at least one biological sample prepared according to the instructions as defined in a) and imaged according to the instructions as defined in b)

2) at least one corresponding reference standard of the at least one

biological sample in c)1)

d) an application description comprising at least

1) information of the preparation protocol as defined in a) and the

instructions for image acquisition as defined in b) a webbased presentation of the application description as defined in d) of a plurality of application packages in the library comprising access request capability - an execution platform providing capabilities of a) viewing digital slides

b) viewing instructions as defined in a), b) and c)

2. The system according to claim 1 , wherein the biological sample is selected from a tissue sample and a body fluid sample.

3. The system according to any of the preceding claims, wherein the biological sample is a tissue section, such as a histological section.

4. The system according to any of the preceding claims, wherein the quantitative digital pathology includes stereology.

5. The system according to any of the preceding claims, wherein the preparation protocol as defined in a) comprises instructions for preparation methods selected from the group consisting of sample collection, sample trimming, sample fixation, sample embedding, sample sectioning, sample staining and sample storage.

6. The system according to any of the preceding claims, wherein the instructions for image acquisition comprises instructions for acquisition selected from the group consisting of magnification, exposure time, modality, resolution, gamma, gain, calibration, scanner, and slides.

7. The system according to any of the preceding claims, wherein one or more auxiliary algorithm is comprised within at least one application package in addition to the image analysis algorithm, such as an auxiliary algorithm for region-of-interest (ROI) detection, and/or removal of artefacts.

8. The system according to any of the preceding claims, wherein reference standard is established by a human as a grade, score, rank, number/count, area, circumference, length, and/or thickness.

9. The system according to any of the preceding claims, wherein the execution platform is provided as a cloud application service.

10. The system according to any of the preceding claims 1 -8, wherein the execution

platform is a deployed application.

1 1 . The system according to any of the preceding claims, wherein the execution platform provides capabilities of viewing the at least one digital slide defined in a)2).

12. The system according to any of the preceding claims, wherein the execution platform further provides capabilities of viewing digital slides positioned on a remote computer system.

13. A method for a user for viewing a digital slide representing structural content of a

biological sample, in a system as defined in any of claims 1 -12 searching the webbased presentation of application descriptions and selecting an application description corresponding to a specific structural content, requesting access through the access request capability, viewing the digital slide(s) corresponding to the selected application description.

14. The method according to claim 13, wherein the digital slide representing the structural content is the at least one digital slide as defined in claim 1 a)2).

15. The method according to claim 13, wherein the digital slide representing the structural content is a slide prepared at user end.

16. The method according to claim 15, wherein the digital slide is prepared according to the instructions as defined in claim 1 a)1) and the image is acquired according to the instructions as defined in claim 1 a)3).

17. The method according to any of the preceding claims 13-16, wherein the execution platform is provided as a cloud application service.

18. The method according to any of the preceding claims 13-16, wherein the execution platform is a deployed application.

19. The method according to any of the preceding claims 13-18, wherein the execution platform further provides capabilities of viewing digital slides positioned on a remote computer system.

20. The method according to any of the preceding claims 15-17, wherein the digital slides are uploaded from the user to the cloud.

21 . The method according to any of the claims 13-20, wherein the biological sample is selected from a tissue sample and a body fluid sample.

22. The method according to any of the preceding claims 13-21 , wherein the biological sample is a tissue section, such as a histological section.

23. The method according to any of the preceding claims 13-22, further including

quantitative digital pathology on the biological sample.

24. The method according to any of the preceding claims 13-23, wherein the quantitative digital pathology includes stereology.