Tutorial: methods for three-dimensional visualization of archival tissue material.

Analysis of three-dimensional patient specimens is gaining increasing relevance for understanding the principles of tissue structure as well as the biology and mechanisms underlying disease. New technologies are improving our ability to visualize large volume of tissues with subcellular resolution. One resource often overlooked is archival tissue maintained for decades in hospitals and research archives around the world. Accessing the wealth of information stored within these samples requires the use of appropriate methods. This tutorial introduces the range of sample preparation and microscopy approaches available for three-dimensional visualization of archival tissue. We summarize key aspects of the relevant techniques and common issues encountered when using archival tissue, including registration and antibody penetration. We also discuss analysis pipelines required to process, visualize and analyze the data and criteria to guide decision-making. The methods outlined in this tutorial provide an important and sustainable avenue for validating three-dimensional tissue organization and mechanisms of disease.

3D single cell scale anatomical map of sex-dependent variability of the rat intrinsic cardiac nervous system.

We developed and analyzed a single cell scale anatomical map of the rat intrinsic cardiac nervous system (ICNS) across four male and three female hearts. We find the ICNS has a reliable structural organizational plan across individuals that provide the foundation for further analyses of the ICNS in cardiac function and disease. The distribution of the ICNS was evaluated by 3D visualization and data-driven clustering. The pattern, distribution, and clustering of ICNS neurons across all male and female rat hearts is highly conserved, demonstrating a coherent organizational plan where distinct clusters of neurons are consistently localized. Female hearts had fewer neurons, lower packing density, and slightly reduced distribution, but with identical localization. We registered the anatomical data from each heart to a geometric scaffold, normalizing their 3D coordinates for standardization of common anatomical planes and providing a path where multiple experimental results and data types can be integrated and compared.

Recommendations for Specimen and Therapy Selection in Colorectal Cancer.

INTRODUCTION: Next-generation sequencing has emerged as a clinical tool for the identification of actionable mutations to triage advanced colorectal cancer patients for targeted therapies. The literature is conflicted as to whether primaries or their metastases should be selected for sequencing. Some authors suggest that either site may be sequenced, whereas others recommend sequencing the primary, the metastasis, or even both tumors. Here, we address this issue head on with a meta-analysis and provide for the first time a set of sensible recommendations to make this determination. METHODS: From our own series, we include 43 tumors from 13 patients including 14 primaries, 10 regional lymph node metastases, 17 distant metastases, and two anastomotic recurrences sequenced using the 50 gene Ion AmpliSeq cancer NGS panel v2. RESULTS: Based on our new cohort and a meta-analysis, we found that ~ 77% of patient-matched primary-metastatic pairs have identical alterations in these 50 cancer-associated genes. CONCLUSIONS: Low tumor cellularity, tumor heterogeneity, clonal evolution, treatment status, sample quality, and/or size of the sequencing panel accounted for a proportion of the differential detection of mutations at primary and metastatic sites. The therapeutic implications of the most frequently discordant alterations (TP53, APC, PIK3CA, and SMAD4) are discussed. Our meta-analysis indicates that a subset of patients who fail initial therapy may benefit from sequencing of additional sites to identify new actionable genomic abnormalities not present in the initial analysis. Evidence-based recommendations are proposed.

A Comprehensive Integrated Anatomical and Molecular Atlas of Rat Intrinsic Cardiac Nervous System.

We have developed and integrated several technologies including whole-organ imaging and software development to support an initial precise 3D neuroanatomical mapping and molecular phenotyping of the intracardiac nervous system (ICN). While qualitative and gross anatomical descriptions of the anatomy of the ICN have each been pursued, we here bring forth a comprehensive atlas of the entire rat ICN at single-cell resolution. Our work precisely integrates anatomical and molecular data in the 3D digitally reconstructed whole heart with resolution at the micron scale. We now display the full extent and the position of neuronal clusters on the base and posterior left atrium of the rat heart, and the distribution of molecular phenotypes that are defined along the base-to-apex axis, which had not been previously described. The development of these approaches needed for this work has produced method pipelines that provide the means for mapping other organs.

Logical Observation Identifiers Names and Codes for Laboratorians.

CONTEXT.­: The Logical Observation Identifiers Names and Codes (LOINC) system is supposed to facilitate interoperability, and it is the federally required code for exchanging laboratory data. OBJECTIVE.­: To provide an overview of LOINC, emerging issues related to its use, and areas relevant to the pathology laboratory, including the subtleties of test code selection and importance of mapping the correct codes to local test menus. DATA SOURCES.­: This review is based on peer-reviewed literature, federal regulations, working group reports, the LOINC database (version 2.65), experience using LOINC in the laboratory at several large health care systems, and insight from laboratory information system vendors. CONCLUSIONS.­: The current LOINC database contains more than 55 000 numeric codes specific for laboratory tests. Each record in the LOINC database includes 6 major axes/parts for the unique specification of each individual observation or measurement. Assigning LOINC codes to a laboratory's test menu should be a defined process. In some cases, LOINC can aid in distinguishing laboratory data among different information systems, whereby such benefits are not achievable by relying on the laboratory test name alone. Criticisms of LOINC include the complexity and resource-intensive process of selecting the most correct code for each laboratory test, the real-world experience that these codes are not uniformly assigned across laboratories, and that 2 tests that may have the same appropriately assigned LOINC code may not necessarily have equivalency to permit interoperability of their result data. The coding system's limitations, which subsequently reduce the potential utility of LOINC, are poorly understood outside of the laboratory.

A Practical Guide to Whole Slide Imaging: A White Paper From the Digital Pathology Association.

CONTEXT.­: Whole slide imaging (WSI) represents a paradigm shift in pathology, serving as a necessary first step for a wide array of digital tools to enter the field. Its basic function is to digitize glass slides, but its impact on pathology workflows, reproducibility, dissemination of educational material, expansion of service to underprivileged areas, and intrainstitutional and interinstitutional collaboration exemplifies a significant innovative movement with far-reaching effects. Although the benefits of WSI to pathology practices, academic centers, and research institutions are many, the complexities of implementation remain an obstacle to widespread adoption. In the wake of the first regulatory clearance of WSI for primary diagnosis in the United States, some barriers to adoption have fallen. Nevertheless, implementation of WSI remains a difficult prospect for many institutions, especially those with stakeholders unfamiliar with the technologies necessary to implement a system or who cannot effectively communicate to executive leadership and sponsors the benefits of a technology that may lack clear and immediate reimbursement opportunity. OBJECTIVES.­: To present an overview of WSI technology-present and future-and to demonstrate several immediate applications of WSI that support pathology practice, medical education, research, and collaboration. DATA SOURCES.­: Peer-reviewed literature was reviewed by pathologists, scientists, and technologists who have practical knowledge of and experience with WSI. CONCLUSIONS.­: Implementation of WSI is a multifaceted and inherently multidisciplinary endeavor requiring contributions from pathologists, technologists, and executive leadership. Improved understanding of the current challenges to implementation, as well as the benefits and successes of the technology, can help prospective users identify the best path for success.

Microphthalmia-associated transcription factor (MiTF): Promiscuous staining patterns in fibrohistiocytic lesions is a potential pitfall.

Microphthalmia-associated transcription factor (MiTF) is used as a marker of melanocytic differentiation. However, MiTF immunoexpression has also been observed in histiocytes, macrophages, smooth muscle cells and fibroblasts, which raise the concern of fibrohistiocytic (FH) lesions being misdiagnosed as melanoma based on MiTF immunoreactivity. MiTF has been known to be positive in FH tumors, but this is the first study evaluating ninety-three fibrohistiocytic neoplasms to understand and delineate the staining pattern of MiTF in these tumors. Ninety-three cases of FH, 30 cases of melanocytic lesions, and 20 miscellaneous cases were studied. The FH cases included benign fibrous histiocytoma (BFH, n = 29), angiofibroma (AF, n = 11), fibromatosis (FM, n = 14), keloid (KE, n = 10), atypical fibroxanthoma (AFX, n = 7), dermal scar (DS, n = 9), dermatofibrosarcoma protuberans (DFSP, n = 12), and pigmented DFSP (Bednar tumor, n = 1). Benign fibrous histiocytoma were sub-categorized into dermatofibroma (n = 15) and epithelioid fibrous histiocytoma (n = 14). The melanocytic lesions included desmoplastic melanoma (DM, n = 8), melanoma in-situ (MIS, n = 5), re-excision-free of melanoma (RFM, n = 10), blue nevus (BN, n = 5), and spitz nevus (SN, n = 3). The miscellaneous category included osteosarcoma (OS, n = 3), pigmented basal cell carcinoma (PBCC, n = 5), spindle cell squamous cell carcinoma (SCA, n = 2), and giant cell tumor of tendon sheath (GCTTS, n = 10). All BFH, AF, AFX, KE, and DS cases showed a positive MiTF staining of variable extent and intensity. MiTF positivity was observed in 86% (n = 12) cases of FM and 17% (n = 2) cases of DFSP. Amongst the miscellaneous category, all cases of PBCC and GCTTS and 50% (n = 1) cases of SCA were immunoreactive for MiTF. All melanocytic lesions were positive for MiTF. None of the OS and pigmented DFSP showed positive labeling. Because of the promiscuity of MiTF labeling, awareness of its pattern in FH proliferations may avoid potential pitfalls in the diagnosis of spindle cell lesions.

Three-dimensional Imaging and Scanning: Current and Future Applications for Pathology.

Imaging is vital for the assessment of physiologic and phenotypic details. In the past, biomedical imaging was heavily reliant on analog, low-throughput methods, which would produce two-dimensional images. However, newer, digital, and high-throughput three-dimensional (3D) imaging methods, which rely on computer vision and computer graphics, are transforming the way biomedical professionals practice. 3D imaging has been useful in diagnostic, prognostic, and therapeutic decision-making for the medical and biomedical professions. Herein, we summarize current imaging methods that enable optimal 3D histopathologic reconstruction: Scanning, 3D scanning, and whole slide imaging. Briefly mentioned are emerging platforms, which combine robotics, sectioning, and imaging in their pursuit to digitize and automate the entire microscopy workflow. Finally, both current and emerging 3D imaging methods are discussed in relation to current and future applications within the context of pathology.

Pathologists' Computer-Assisted Diagnosis: A Mock-up of a Prototype Information System to Facilitate Automation of Pathology Sign-out.

CONTEXT: - Pathologists' computer-assisted diagnosis (pCAD) is a proposed framework for alleviating challenges through the automation of their routine sign-out work. Currently, hypothetical pCAD is based on a triad of advanced image analysis, deep integration with heterogeneous information systems, and a concrete understanding of traditional pathology workflow. Prototyping is an established method for designing complex new computer systems such as pCAD. OBJECTIVE: - To describe, in detail, a prototype of pCAD for the sign-out of a breast cancer specimen. DESIGN: - Deidentified glass slides and data from breast cancer specimens were used. Slides were digitized into whole-slide images with an Aperio ScanScope XT, and screen captures were created by using vendor-provided software. The advanced workflow prototype was constructed by using PowerPoint software. RESULTS: - We modeled an interactive, computer-assisted workflow: pCAD previews whole-slide images in the context of integrated, disparate data and predefined diagnostic tasks and subtasks. Relevant regions of interest (ROIs) would be automatically identified and triaged by the computer. A pathologist's sign-out work would consist of an interactive review of important ROIs, driven by required diagnostic tasks. The interactive session would generate a pathology report automatically. CONCLUSIONS: - Using animations and real ROIs, the pCAD prototype demonstrates the hypothetical sign-out in a stepwise fashion, illustrating various interactions and explaining how steps can be automated. The file is publicly available and should be widely compatible. This mock-up is intended to spur discussion and to help usher in the next era of digitization for pathologists by providing desperately needed and long-awaited automation.

The coming paradigm shift: A transition from manual to automated microscopy.

The field of pathology has used light microscopy (LM) extensively since the mid-19(th) century for examination of histological tissue preparations. This technology has remained the foremost tool in use by pathologists even as other fields have undergone a great change in recent years through new technologies. However, as new microscopy techniques are perfected and made available, this reliance on the standard LM will likely begin to change. Advanced imaging involving both diffraction-limited and subdiffraction techniques are bringing nondestructive, high-resolution, molecular-level imaging to pathology. Some of these technologies can produce three-dimensional (3D) datasets from sampled tissues. In addition, block-face/tissue-sectioning techniques are already providing automated, large-scale 3D datasets of whole specimens. These datasets allow pathologists to see an entire sample with all of its spatial information intact, and furthermore allow image analysis such as detection, segmentation, and classification, which are impossible in standard LM. It is likely that these technologies herald a major paradigm shift in the field of pathology.

Exploring virtual reality technology and the Oculus Rift for the examination of digital pathology slides.

BACKGROUND: Digital slides obtained from whole slide imaging (WSI) platforms are typically viewed in two dimensions using desktop personal computer monitors or more recently on mobile devices. To the best of our knowledge, we are not aware of any studies viewing digital pathology slides in a virtual reality (VR) environment. VR technology enables users to be artificially immersed in and interact with a computer-simulated world. Oculus Rift is among the world's first consumer-targeted VR headsets, intended primarily for enhanced gaming. Our aim was to explore the use of the Oculus Rift for examining digital pathology slides in a VR environment. METHODS: An Oculus Rift Development Kit 2 (DK2) was connected to a 64-bit computer running Virtual Desktop software. Glass slides from twenty randomly selected lymph node cases (ten with benign and ten malignant diagnoses) were digitized using a WSI scanner. Three pathologists reviewed these digital slides on a 27-inch 5K display and with the Oculus Rift after a 2-week washout period. Recorded endpoints included concordance of final diagnoses and time required to examine slides. The pathologists also rated their ease of navigation, image quality, and diagnostic confidence for both modalities. RESULTS: There was 90% diagnostic concordance when reviewing WSI using a 5K display and Oculus Rift. The time required to examine digital pathology slides on the 5K display averaged 39 s (range 10-120 s), compared to 62 s with the Oculus Rift (range 15-270 s). All pathologists confirmed that digital pathology slides were easily viewable in a VR environment. The ratings for image quality and diagnostic confidence were higher when using the 5K display. CONCLUSION: Using the Oculus Rift DK2 to view and navigate pathology whole slide images in a virtual environment is feasible for diagnostic purposes. However, image resolution using the Oculus Rift device was limited. Interactive VR technologies such as the Oculus Rift are novel tools that may be of use in digital pathology.

Digital pathology and anatomic pathology laboratory information system integration to support digital pathology sign-out.

BACKGROUND: The adoption of digital pathology offers benefits over labor-intensive, time-consuming, and error-prone manual processes. However, because most workflow and laboratory transactions are centered around the anatomical pathology laboratory information system (APLIS), adoption of digital pathology ideally requires integration with the APLIS. A digital pathology system (DPS) integrated with the APLIS was recently implemented at our institution for diagnostic use. We demonstrate how such integration supports digital workflow to sign-out anatomical pathology cases. METHODS: Workflow begins when pathology cases get accessioned into the APLIS (CoPathPlus). Glass slides from these cases are then digitized (Omnyx VL120 scanner) and automatically uploaded into the DPS (Omnyx(®) Integrated Digital Pathology (IDP) software v.1.3). The APLIS transmits case data to the DPS via a publishing web service. The DPS associates scanned images with the correct case using barcode labels on slides and information received from the APLIS. When pathologists remotely open a case in the DPS, additional information (e.g. gross pathology details, prior cases) gets retrieved from the APLIS through a query web service. RESULTS: Following validation of this integration, pathologists at our institution have signed out more than 1000 surgical pathology cases in a production environment. Integration between the APLIS and DPS enabled pathologists to review digital slides while simultaneously having access to pertinent case metadata. The introduction of a digital workflow eliminated costly manual tasks involving matching of glass slides and avoided delays waiting for glass slides to be delivered. CONCLUSION: Integrating the DPS and APLIS were instrumental for successfully implementing a digital solution at our institution for pathology sign-out. The integration streamlined our digital sign-out workflow, diminished the potential for human error related to matching slides, and improved the sign-out experience for pathologists.

International Telepathology: Promises and Pitfalls.

Innovative technologies for digital imaging and telecommunications are changing the way we deliver health care. Telepathology collaborations are one example of how delivering remote pathology services to patients can benefit from leveraging this change. Over the years, several academic and commercial teleconsultation networks have been established. Herein, we review the landscape of these international telepathology efforts and highlight key supportive factors and potential barriers to successful cross-border collaborations. Important features of successful international telepathology programs include efficient workflows, dedicated information technology staff, continuous maintenance, financial incentives, ensuring that all involved stakeholders are satisfied, and value-added clinical benefit to patient care. Factors that plague such telepathology operations include legal/regulatory issues, sustainability, and cultural and environmental issues. Pathologists, vendors and laboratory accreditation agencies will need to embrace and capitalize on this new paradigm of international telepathology accordingly.

Overview of Telepathology.

Telepathology is the practice of remote pathology using telecommunication links to enable the electronic transmission of digital pathology images. Telepathology can be used for remotely rendering primary diagnoses, second opinion consultations, quality assurance, education, and research purposes. The use of telepathology for clinical patient care has been limited mostly to large academic institutions. Barriers that have limited its widespread use include prohibitive costs, legal and regulatory issues, technologic drawbacks, resistance from pathologists, and above all a lack of universal standards. This article provides an overview of telepathology technology and applications.

Overview of Telepathology.

Telepathology is the practice of remote pathology using telecommunication links to enable the electronic transmission of digital pathology images. Telepathology can be used for remotely rendering primary diagnoses, second opinion consultations, quality assurance, education, and research purposes. The use of telepathology for clinical patient care has been limited mostly to large academic institutions. Barriers that have limited its widespread use include prohibitive costs, legal and regulatory issues, technologic drawbacks, resistance from pathologists, and above all a lack of universal standards. This article provides an overview of telepathology technology and applications.