2016

‘How to Go Digital in Pathology’ LabPON whitepaper (made possible by Philips)

Introduction

Digital microscopy (pathology) is gaining interest worldwide. For instance, in Scandinavia (1) (2), and in Canada - through the Canadian Association of Pathologists - there are now established guidelines for telepathology service for anatomic pathology (3). Some Dutch pathology laboratories in the Netherlands are leaders in the transition to work digitally. Laboratory for Pathology East Netherlands (LabPON) is one of the first in the world to make the step to implement a fully digitized histopathological clinical diagnostics workflow.

In this paper, we provide insight into the transition to a fully digitized histopathological clinical workflow in a pathology laboratory. We believe our experience can help other laboratories complete this process smoothly and as costeffectively as possible. We describe our transition in the following terms: business vision, logistics, technical aspects, and ergonomics. The implementation in our laboratory was handled gradually and effectively.

LabPON is one of the largest pathology laboratories in The Netherlands and in Europe. It is an independent laboratory with 115 employees of whom 18 are pathologists, handling more than 55,000 histological requests per year, with a turnaround time of 3 to 5 working days. The mission statement of LabPON is to provide fast, efficient pathology service of the highest quality to physicians and hospitals. We realize this is thanks to our clear corporate vision that allows creativity to bloom without interfering with company operations.

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‘Bidirectional integration in digital pathology’ LabPON whitepaper (made possible by Philips)

Introduction

Digital pathology solutions speed up and simplify the access to histopathology. Comprehensive bidirectional integration with a Laboratory Information Management System (LIMS) is essential to optimally benefit from the improvement in efficiency and quality that digital pathology solutions offer.

LabPON (Hengelo, The Netherlands), being one of the first clinical pathology laboratories to have transitioned fully to digital diagnosis, emphasizes the need for bidirectional integration.

“In transferring from conventional microscopic evaluation of slides to digital viewing we have found a comprehensive integration our Image management System (IMS) and our Laboratory Information Management System (LIMS) essential for patient safety. Moreover, good integration results in further time savings and higher accuracy in the overall process”

At LabPON, every day material arrives from three hospitals and from regional general practitioners. After processing of the material and staining, the slides are dispatched to all pathologists present that day and a diagnosis is made and reported. Digitization of slides was implemented to increase efficiency and reduce the shortcomings of a traditional analog pathology work flow. The Finalist LIMS was already used for a long time to handle specimens in the lab and to track the steps in processing from arrival and registration of a case up until sending of the final report to the requesting physician. To ensure efficient and secure data handling, an adjusted workflow was essential. LabPON was able to create a workflow in which LIMS and IMS are communicating and sharing information bi-directionally. In practice, the use of barcodes ensures that tissue is traceable during the entire route. Every step in the process is logged;from the entry of patient data and clinical information until the authorization of the final report. The barcode generated by the Finalist LIMS is read by the Philips Ultra Fast Scanner (UFS). The barcode information and the image data as yielded by the UFS are coupled by the Philips Image Management System (IMS) server. The IMS in turn exchanges information with the LIMS server. The UFS and IMS are part of Philips IntelliSite Pathology Solution, an open platform for digital pathology. The information transferred to the IMS is shown both in the work list of the LIMS and the IMS case viewer as as well as on the individual slide images within the case. The work list contains all cases that a pathologist needs to review. The system will trigger the LIMS to show the matching report of the cases open in IMS, preventing mix-ups while reporting. It is possible to start with opening a case viewer and reporting from either the LIMS or the IMS. Relevant patient information is shared between the LIMS and the IMS viewer. Cases handled by IMS are checked and evaluated by LIMS and vice versa. The full and bidirectional integration of both systems warrants accurate matching of cases. Any change in one system will lead to appropriate matching in the other.

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Philips’ iSyntax for Digital Pathology

Introduction

Digital pathology Whole Slide Images (WSI) are gigapixel sized images, that need to be created in less than a minute on a fast whole slide scanner such as the Philips Ultra Fast Scanner.

Additionally a suitable image format for WSI needs to support fast zooming and panning. Currently the most common image format used for storing whole slide images is the TIFF image format with the JPEG image compression method. While this satisfies the speed requirements both for image compression as well as for fast image decompression in web applications, the JPEG compression standard is a consumer grade compression method, and not optimal for medical grade images. In particular the decimation of the color resolution and the low dynamic range of 8 bit per channel and the limitation to three color channels is problematic.

Additionally JPEG is not an hierarchical image compression method, meaning that it is not possible to quickly retrieve a lower resolution representation of a subregion of an image. This problem is typically overcome by storing multiple lower resolution versions of the image in the same TIFF file - often referred to as the pyramid TIFF format; this however results in a 30% larger image file at the same image quality level, or in a lower image quality at the same file size. The limitations of the now 24 year old JPEG standard are well known, and in the year 2000 the JPEG group created JPEG 2000 to supersede the JPEG standard. JPEG 2000 takes away most if not all limitations in JPEG that make it less suitable for medical grade images: it supports high dynamic range images (up to 32bit per channel), it does not force a color resolution decimation, and it supports multiple color channels. In addition, because it is a wavelet based compression method it is inherently a hierarchical image format which allows for fast retrieval of lower resolution versions of the original image without the need for storing the redundant pyramid of lower resolution versions of the image as required by the JPEG TIFF image format.

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Philips IntelliSite Open Pathology Platform

Introduction

Since the development of modern anatomical pathology as a medical discipline in the 19 Century it has been uniquely bound to the location of the specimen, requiring direct (microscopic) inspection of the biopsy.

Today Digital Pathology is breaking those bonds, allowing the practice of anatomical pathology anywhere, independent from the physical location of the biopsy and thus the patient. Remarkably enough, the coming of age of high speed and high quality digital microscope slide scanners coincides with the success of globe spanning digital sharing and collaboration networks: the collaborative encyclopedia WikiPedia, GitHub for collaborative software development, Office 365 for business office software. Digital Pathology is enabled by these technology revolutions to convert local pathology labs into globe-spanning virtual pathology networks. In this white paper I will share with you Philips’ vision on the future of collaborative digital pathology, and explain why it needs an open platform approach to succeed.

Digital Pathology needs to bring mobility and flexibility to the pathologist, so that he or she can work from anywhere, and present, share and participate fully in multidisciplinary team meetings, tumor-boards, and conferences. All without any concessions to patient safety and privacy. A fatal -- though perhaps understandable -- mistake would be to address this by simply replacing the current anatomical pathology’s practice of moving physical tissue blocks and slides from A to B by a practice of moving digital image files of microscopy slides from A to B. There are three reasons why enabling collaboration in digital pathology by sharing whole slide image files will not be productive:

1. Security, privacy and patient consent are impossible to guarantee when patient records, such as whole slide images, are exchanged as files via network shares or portable media.
2. Mobile workers require data that can be accessed from anywhere. The mobile software paradigm does not, and will never, support local storage of large data sets such as medical images.
3. The sheer sizes of digital whole slide images prevents effectively storing, sharing and analyzing on anything but server grade hardware that does typically not allow file based interactions.

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Robust detection of RNA biomarkers for drug safety assessment in preclinical animal models by fully automated RNAscope® 2.5 LS Assay

Preclinical drug safety assessment in animal models is used to evaluate the pathological effects induced by novel therapeutic agents. Here we present the use of the RNAscope^® 2.5 LS Assay for the evaluation of biomarkers in tissues from three preclinical animal models. In this study we:

• Identify optimal pretreatment conditions for different tissues in different species
• Provide recommendations for control gene selection for tissue qualification
• Detect specific RNA markers in various FFPE tissues from multiple species

Abstract

Robust assays to evaluate biomarkers in tissue are needed for preclinical safety assessment and toxicity studies. Here we present the application of the fully automated RNAscope^® 2.5 LS Assay on Leica BOND RX for RNA in situ hybridization in formalin-fixed paraffin-embedded (FFPE) tissues from three commonly used animal models (rat, cynomolgus monkey, and dog). We demonstrate robust assay performance with high signal-tonoise ratio and well-maintained morphology in 25 different tissues from each species. Based on these tests, we provide recommendations for proper control gene for sample qualification of each tissue type, as well as optimal pretreatment protocol selection. For specific target RNA markers, we successfully detected cell type specific markers such as CD31 (PECAM1) and CD68, proliferation marker Ki-67 (MKI67), and cell cycle marker Cyclin E1 (CCNE1), as well as apoptosis-related molecules Puma (BBC3), Fas (CD95), and DR5 (TNFRSF10B). This study demonstrates that the RNAscope^® 2.5 LS Assay can be an attractive platform for biomarker analysis in tissues for preclinical safety assessment and general animal studies.

Introduction

Preclinical drug safety assessment in animal models has been well established as a routine laboratory practice to evaluate the pathological alterations induced by novel therapeutic agents1. This preliminary evaluation serves a major role in the development of new treatments prior to trials in humans. Histopathological techniques have been traditionally applied for general morphological evaluation by hematoxylin and eosin (H&E) stain, and also for evaluation of specific biomarkers by immunohistochemistry (IHC). IHC assay has been commonly applied to assess therapeutic targets and toxicity-related biomarkers. However, consistent and systematic application of IHC techniques has been hindered by inconsistent performance of various antibody clones, time-consuming antibody development and validation, and general lack of reagents for some animal models. RNA in situ hybridization (ISH) technology presents an attractive alternative method for pathological evaluation of biomarkers in tissues from various preclinical animal models, because nucleic acid-based probes specific for any biomarkers associated with drug toxicity or mechanism can be developed and validated rapidly. The RNAscope^® technology, an advanced platform for in situ RNA detection, enables detection of almost any RNA biomarker with single-molecule detection sensitivity and high specificity in formalin-fixed paraffin-embedded (FFPE) tissues². It provides a universal solution to characterize tissue distribution of drug targets and biomarkers in a highly specific and sensitive manner, without the need to wait for antibody development and validation. The RNAscope^® assay can be performed in fully automated staining systems, including Ventana Discovery XT, Ventana Discovery ULTRA, and Leica BOND RX instruments. The assay allows visualization of each individual RNA molecule as a punctate dot under a standard bright field microscope. The RNA dots can be quantified by counting the number of signal dots in individual cells, either manually or by image analysis tools, including HALOTM (Indica Labs) and SpotStudio^TM (ACD) software. In this study, we demonstrate the feasibility of evaluating RNA biomarkers in 25 types of tissues from three commonly used animal models using the RNAscope^® 2.5 LS Reagent Kit-BROWN on the Leica BOND RX instrument. Robust RNA detection was achieved following a standard protocol in almost all of the tissues tested, with minor alterations to the prestaining conditions for a few tissues. Here, we identified the threshold of pretreatment needed for different tissue types. We also provide recommendations for control probes to be applied for tissue qualification, and present the evaluation of RNA biomarkers including cell type specific markers (CD68 and endothelial marker PECAM1), proliferation marker Ki-67 (MKI67), and cell cycle marker Cyclin E1 (CCNE1). Overall, our study shows that the fully automated RNAscope^® 2.5 LS Assay is capable of detecting a broad range of RNA targets in all major tissue types with little to no optimization needed, and thus well suited for the histopathological evaluation of biomarkers in the assessment of drug-derived toxicity in various tissues and animal models.

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Cellular localization of RNA expression in central and peripheral nervous system using RNAscope® Technology

Detect your RNA of interest in the central and peripheral nervous system with ease

• Detection of virtually any gene in any genome in any tissue with high sensitivity and specificity
• Visualize RNA expression and distribution with morphological context
• Uncover lncRNAs and other targets for which antibodies cannot detect

Detection of RNA in nervous system using RNAscope® Technology

Neuroscience is one of the fastest growing research fields, studying the structural and functional organization and the development of the central and peripheral nervous system from the molecular and cellular level to the systems level.

One challenge in the neuroscience field is the numerous cell types in the central nervous system (CNS), many of which remain to be identified and characterized at the molecular level.

RNAscope^® in situ hybridization (ISH) technology enables cell-specific localization of RNA transcripts precisely in the tissue to identify markers associated with neuronal and synaptic molecular pathways and to characterize specific cell types such as neuronal and glial cells.

The RNAscope^® assay can be used to:

• Identify, characterize, and localize mRNAs in the central and peripheral nervous systems
• Detect mRNA expression in axons and dendrites
• Detect mRNA in the nervous system when no reliable antibodies are available
• Identify long non-coding RNA in neuronal and glial cells
• Detect mRNA in primary neuronal cell cultures

RNAscope^® ISH technology provides a unique detection method for neuronal and glial cells markers that is both highly sensitive and specific for detection of any gene expressed in any neuronal and glial cell¹. Because of its proprietary “double Z” probe, RNAscope^® technology is a powerful tool to detect any gene, especially low abundance targets. And not only can RNAscope^® technology be used to discern if your gene of interest is in a neuronal or glial cell, but it can also show its subcellular localization. The multiplexing capabilities of RNAscope^® assay allows for detection of multiple markers at once, enabling exquisite characterization of cell populations within the nervous system as well as detection of both neuronal and glial cell markers and the signals these cells produce. RNAscope® technology also helps solve the problem when no reliable antibodies exist for your gene of interest and allows you to visualize and quantify for virtually any gene from any genome in any tissue.

FIGURE 1. Detection of Otx2 mRNA in mouse cerebellum FFPE tissue with the RNAscope® 2.5 HD Reagent Kit - BROWN.

FIGURE 2. Mash1 mRNA expression in mouse brain tissue, RNA in situ hybridization (ISH) using RNAscope® 2.5 HD Reagent Kit - BROWN.

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Detection of immune cell checkpoint and functional markers in the tumor microenvironment by the RNA in situ hybridization RNAscope® assay

The RNAscope® assay is a unique RNA ISH technology that identifies RNA expression at the single cell level with morphological context. Here we present the use of RNAscope® for the detection of RNA targets in the tumor microenvironment (TME) that are involved in tumor immunology and immunotherapy:

• Checkpoint markers
• Immune cell markers
• Cytokines and chemokines

Detection of these RNA targets with the RNAscope® assay can aid in determining:

• Localization of specific immune cell types (i.e., cytotoxic lymphocytes and regulatory T cells) in the TME
• Spatial relationships between different cell types in the TME
• Characterization of secreted proteins (i.e., cytokines and chemokines)
• Evaluation of immune function in TME beyond enumeration of tumor infiltrating lymphocytes

Introduction

The field of cancer immunotherapy has expanded rapidly in recent years with promising clinical results by immune checkpoint inhibitors and other therapeutic approaches such as cancer vaccines and chimeric antigen receptor (CAR) therapy. Despite the dramatic and durable responses seen in many patients, our understanding of the immune response to cancer is still limited, and we cannot reliably predict who will or will not benefit from these new interventions. To better stratify patients for immunotherapy treatments, the series of events and biomarkers involved in the cancer‑immunity cycle need to be better understand^(1-3). In addition, spatially mapped expression data at the single-cell level is crucial to understanding the cellular organization and cell-to-cell interactions in the tumor and its complex microenvironment (TME).

RNAscope^® is a unique RNA ISH technology that provides single-cell gene expression resolution with spatial and morphological context. The RNAscope^® assay detects mRNA and long noncoding RNAs in fresh frozen, fresh fixed, and formalin-fixed paraffin-embedded (FFPE) cells and tissues by utilizing a unique double Z probe design and signal amplification strategy that allows for visualization of target RNA as a single dot, where each dot is an individual RNA molecule⁽⁴⁾. The key benefit of the RNAscope^® strategy is high sensitivity due to its amplification method and high specificity because of the double Z probe design, leading to a high signal-to-noise ratio in many tissues. In this report we examined 50 selected immune checkpoint and functional markers.

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The Betsi Cadwaladr University Health Board (BCUHB) is the largest single health organisation in Wales, and is responsible for providing community, hospital and mental health services, and overseeing primary health care services for a population of around 680,000 people in North Wales, together with providing some services to residents of North Powys and parts of Cheshire and Shropshire. The services are provided from three acute hospitals: Ysbyty Gwynedd (YG) in Bangor; Glan Clwyd Hospital near Rhyl (YGC); and Wrexham Maelor Hospital (YMW), along with a network of community hospitals, health centres, clinics, mental health units and community team bases. We employ approximately 16,000 staff, and also coordinate the work of 191 general practitioner (GP) practices, and National Health Service (NHS) services provided by dentists, opticians and pharmacists across the region. BCUHB was established on 1 October 2009 as a part of the Welsh Government One Wales reform programme for the NHS in Wales, bringing together three acute and several community hospitals, along with six primary care commissioning area units as a single provider. The reconfiguration provided great opportunity for closer working together by multiple organisations in the north of Wales to provide best possible and specialist care for our patients. The greatest constraint, similar to any mixed urban/rural population, is geographical – presenting confounding issues in respect of having adequate critical mass to support sub-specialist care provision while maintaining acceptable access times for the majority of patients.

For Pathology services across North Wales, a single managed service was created to replace the three individual hospitalbased departments. With the relatively low requirement for direct patient access to laboratory-based Pathology services, the opportunity existed to re-provide these services in a more specialist, concentrated form. During the period 2010–2015, several business cases were approved and implemented, to support this vision, providing new/refurbished premises, specialist laboratory equipment and IT technologies to improved both quality and performance of essential investigations and tests.

Cellular pathology in North Wales

For Cellular Pathology services, the outcome of this major reconfiguration was a merge of the three small cellular pathology departments into one single managed central laboratory based in Glan Clwyd Hospital site near Rhyl; a new, purpose-built laboratory and accommodation for up to 12 medical staff and their secretarial teams. The service operates principally from the Glan Clwyd site, with currently sample reception, mortuary, and minimal consultant/ secretarial accommodation and facilities remaining at the Ysbyty Wrexham/Ysbyty Gwynedd sites. The new single Cellular Pathology service annually processes:

• 44,000 histopathology requests
• 5000 non-cervical cytology requests
• 50,000 cervical cytology requests.

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Routine integration of fully automated image analysis of Kreatech CLL FISH Probes, using the CytoVision GSL Scanning System

Executive Summary

The automation of FISH slide scoring in a high throughput fashion has long been a goal for cytogenetic analysis. The development of CytoVision automated scanning and image analysis has enabled realization of this aspiration. In the current study, automation was utilized across seven assays, in combination with a CLL menu of Kreatech FISH probes. The results demonstrate that the combination of Kreatech FISH probes with a fully automated, unsupervised analysis can successfully be used to classify patient samples according to known CLL-associated genomic abnormalities. The overall comparative analysis per CLL probe set, between manually scored slides and slides analyzed using automation and unsupervised analysis, showed at least 94% concordance, which increased to 100% following subsequent review. Overall, the hands-on time was drastically reduced by 88% (48 hr 45 min) and a permanent record for each sample automatically created. The full automation of the image analysis process using the CytoVision GSL-120 scanning system demonstrates the versatility and effectiveness of the system to be easily integrated into high throughput, routine FISH analysis.

Summary

B-cell chronic lymphocytic leukemia (CLL) is the most common leukemia in adults, with a median survival of ~9 years (1). The disease is characterized by a highly variable clinical course. Fluorescence in situ hybridization (FISH) has been shown to detect genomic abnormalities in over 80% of CLL cases (2, 3). A menu of seven specific molecular biomarkers detectable using FISH assays has been established within the Leica Biosystems’ repeat-free Kreatech FISH portfolio to predict clinical outcomes per patient. These seven DNA FISH probes have been selected to detect prevalent genetic aberrations found in B-cell CLL with known diagnostic value. The most common recurrent chromosomal abnormalities in CLL include deletion of 13q14 in 40 - 55% of cases, trisomy 12 in 16 - 20% of cases and deletion of 11q22 and deletion of 17p13 in 14 - 20% and 7 - 11% of cases respectively (4, 5, and 6). These genomic aberrations are used to group patients into the Döhner categories, providing prognostic information on each patient subgroup (7, 8, 9, 10). Five prognostic categories have been defined in a hierarchical model. Those with 17p13 deletions show the worst prognosis, followed by those with 11q22 deletions. Patients with trisomy 12 and those with normal karyotypes show intermediate survival. Patients with 13q14 deletions as the sole abnormality had the longest estimated survival times (7, 8). The automated analysis of common FISH tests in routine cytogenetic laboratories has many potential benefits. Obvious benefits include a more efficient workflow, freeing up valuable laboratory staff time and increased consistency of scoring. In addition, the ability to work more ergonomically and without the need for a darkroom would be attractive for many laboratories and personnel. Moving the analysis away from manual microscopes also has the potential to lower costs, and utilizing on the screen analysis could simplify staff training. Automated routine FISH analysis requires slide preparation to be of a consistent high quality. At the Leica Biosystems’ Amsterdam site a range of high-quality FISH probes are manufactured for commercial sale. During manufacture, the CytoVision GSL-120 platform is used as part of the quality control process. In this study we investigated the use of CytoVision GSL-120 for a set of common genomic aberrations associated with CLL, compared with typical manual dual scoring in order to investigate the potential for use of such analysis in a routine laboratory workflow. This study was designed and conducted to compare fully automated scanning and unsupervised image analysis of patient samples versus manual signal enumeration per probe set within the CLL menu of Kreatech FISH probes.

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Automating the cytogenetics process

The cytogenetics laboratory at the Regional Hospital of Treviso performs tests in different fields of clinical medicine including cancer cytogenetics (onco-haematology and solid tumours) and postnatal tests to detect constitutional abnormalities. The laboratory analyses approximately 2200 cases per year.

Techniques employed and clinical indications

The main techniques employed are: karyotyping/chromosome analysis; fluorescence in situ hybridisation (FISH); and comparative genomic hybridisation (array-CGH).

Karyotyping

Classical cytogenetic analysis is carried out on peripheral blood, skin and bone marrow samples to detect constitutional and acquired abnormalities. The clinical indications for investigation of constitutional karyotype are:

• Primary or secondary amenorrhoea or premature menopause
• Sperm abnormalities, such as azoospermia or severe oligospermia
• Clinically significant abnormal growth
• Ambiguous genitalia
• Abnormal clinical phenotype or dysmorphism
• Multiple congenital abnormalities
• Intellectual disability or developmental delay;
• Suspected deletion/duplication syndrome
• A malformed foetus or stillbirth of unknown aetiology
• Third and subsequent consecutive miscarriages.

Couples with:

• Chromosome abnormality or unusual variant detected at prenatal diagnosis
  
• Unbalanced chromosome abnormality in the products of conception
  
• Child with a chromosome abnormality
  
• Infertility of unknown aetiology.

The clinical indications for investigation of acquired karyotype include: acute leukaemia; myelodysplasia; chronic myelogenous leukaemia; chronic myeloproliferative neoplasms; malignant lymphoma and lymphoproliferative disorders; and chronic lymphocytic leukaemia. Side volume for karyotyping is 850 cases/year.

FISH

FISH is employed on cytological samples (peripheral blood and bone marrow) and histological sections of solid tumours. Side volume for FISH is 1200 cases/year.

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