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Multiparametric fluorescence imaging through CODEX allows the simultaneous imaging of many biomarkers in a single tissue section. While the digital fluorescence data thus obtained can provide highly specific characterizations of individual cells and microenvironments, the images obtained are different from those usually interpreted by pathologists (i.e., hematoxylin and eosin [H&E] slides and 3,3′-diaminobenzidine-stained immunohistochemistry slides). Having the fluorescence data plus coregistered H&E or similar data could facilitate the adoption of multiparametric imaging into regular workflows, as well as facilitate the transfer of algorithms and machine learning previously developed around H&E slides. Since commercial CODEX instruments do not produce H&E-like images by themselves, we developed a staining protocol and associated image processing to make “virtual H&E” images that can be incorporated into the CODEX workflow. While there are many ways to achieve virtual H&E images, including the use of a fluorescent nuclear stain and tissue autofluorescence to simulate eosin staining, we opted to combine fluorescent nuclear staining (through 4′,6-diamidino-2-phenylindole) with actual eosin staining. We also output images derived from fluorescent nuclear staining and autofluorescence images for additional evaluation.

Background: Whole-slide images (WSI) are produced by a high-resolution scanning of pathology glass slides. There are a large number of whole-slide imaging scanners, and the resulting images are frequently larger than 100,000 × 100,000 pixels which typically image 100,000 to one million cells, ranging from several hundred megabytes to many gigabytes in size. Aims and Objectives: Provide HTTP access over the web to Whole Slide Image tiles that do not have localized tiling servers but only basic HTTP access. Move all image decode and tiling functions to calling agent (ImageBox). Methods: Current software systems require tiling image servers to be installed on systems providing local disk access to these images. ImageBox2 breaks this requirement by accessing tiles from remote HTTP source via byte-level HTTP range requests. This method does not require changing the client software as the operation is relegated to the ImageBox2 server which is local (or remote) to the client and can access tiles from remote images that have no server of their own such as Amazon S3 hosted images. That is, it provides a data service [on a server that does not need to be managed], the definition of serverless execution model increasingly favored by cloud computing infrastructure. Conclusions: The specific methodology described and assessed in this report preserves normal client connection semantics by enabling cloud-friendly tiling, promoting a web of http connected whole-slide images from a wide-ranging number of sources, and providing tiling where local tiling servers would have been otherwise unavailable.

Introduction: Successful implementation of whole slide imaging (WSI) for routine clinical practice has been accomplished in only a few pathology laboratories worldwide. We report the transition to an effective and complete digital surgical pathology workflow in the pathology laboratory at Cannizzaro Hospital in Catania, Italy. Methods: All (100%) permanent histopathology glass slides were digitized at ×20 using Aperio AT2 scanners. Compatible stain and scanning slide racks were employed to streamline operations. eSlide Manager software was bidirectionally interfaced with the anatomic pathology laboratory information system. Virtual slide trays connected to the two-dimensional (2D) barcode tracking system allowed pathologists to confirm that they were correctly assigned slides and that all tissues on these glass slides were scanned. Results: Over 115,000 glass slides were digitized with a scan fail rate of around 1%. Drying glass slides before scanning minimized them sticking to scanner racks. Implementation required introduction of a 2D barcode tracking system and modification of histology workflow processes. Conclusion: Our experience indicates that effective adoption of WSI for primary diagnostic use was more dependent on optimizing preimaging variables and integration with the laboratory information system than on information technology infrastructure and ensuring pathologist buy-in. Implementation of digital pathology for routine practice not only leveraged the benefits of digital imaging but also creates an opportunity for establishing standardization of workflow processes in the pathology laboratory.

Background: With the adoption of a completely electronic workflow by several journals and the advent of telepathology, digital imaging has become an integral part of every scientific research. However, manipulating digital images is very easy, and it can lead to misinterpretations. Aim: To analyse the impact of manipulating digital images on their diagnosis. Design: Digital images were obtained from Papanicolaou-stained smears of dysplastic and normal oral epithelium. They were manipulated using GNU Image Manipulation Program (GIMP) to alter their brightness and contrast and color levels. A Power Point presentation composed of slides of these manipulated images along with the unaltered originals arranged randomly was created. The presentation was shown to five observers individually who rated the images as normal, mild, moderate or severe dysplasia. Weighted k statistics was used to measure and assess the levels of agreement between observers. Results: Levels of agreement between manipulated images and original images varied greatly among observers. Variation in diagnosis was in the form of overdiagnosis or under-diagnosis, usually by one grade. Conclusion: Global manipulations of digital images of cytological slides can significantly affect their interpretation. Such manipulations should therefore be kept to a minimum, and avoided wherever possible.

There are definite lessons to be learned from the digital radiology experience as pathology transitions toward more widespread use of digital images for diagnostic purposes. While there are parallels between digital radiology and digital pathology in terms of work flow gains and losses, there are also important differences like pathology’s need for color images and the large files that are created when slides are digitized as whole slide images (WSI). The paper by Patterson et al,[1] points out that the adoption of digital imaging by pathologists has been slower than that encountered with filmless radiology. To explore this issue deeper, the authors conducted semi-structured interviews with radiologists and pathologists and looked at the adoption of other health information technologies. Their results indicated that pathologists have a lower opinion of the overall performance of digital systems than their radiologist counterparts. Specific issues of concern included: differences in magnification and image scale as compared to light microscopy, large file sizes and data management, longer time to review individual slides and an inability to focus on folded or uneven areas of tissue. In addition, hardware and software costs, information technology (IT) support, LIS integration, regulatory issues and lack of standards or best practice guidelines also represent key obstacles to the more widespread adoption of digital pathology. Even if the issues around regulations, standards, cost and infrastructure were to suddenly vanish, the perception of inferior performance of WSI systems by pathologists will prevent more rapid adoption. The issue of inability to adjust focus on digital images on folded or uneven areas of tissue highlights the dependence of current-state digital pathology systems on good quality histology. It also raises obvious concerns about diagnostic accuracy. As pointed out by one of the pathologists in the study by Patterson et al, inferior performance (assuming this refers to diagnostic accuracy) on even a small percentage of cases could have major implications for high risk diagnoses. The importance of this point as a barrier to adoption cannot be overemphasized. As pointed out in a recent Scientific American article,[2] the practice of pathology has been based on glass slides and light microscopes for over 100 years and digital pathology systems represent disruptive technology. The prospect of such a major change will naturally cause pathologists to be reluctant about adopting a technology that could both slow them down and introduce the possibility of diagnostic error. Having said this, it must also be acknowledged that WSI technology is steadily improving and vendors are acutely aware of the need for outstanding image quality and faster scanning speeds. Validation studies performed in a variety of institutions and settings using these improved systems will play a critical role in determining the rate of adoption. Whether these studies are based on histologic feature recognition, diagnostic concordance or a combination of the two, the results must demonstrate equivalence between WSI systems and light microscopy if a sense of confidence is to develop across the pathology community as a whole. The survey by Patterson et al, identified many facilitators for the adoption of digital pathology. These included the benefits of using digital pathology platforms for medical student and pathology resident training, continuing medical education and tumor boards. All of these activities share a theme of increasing the exposure of the pathologists to this technology using optimized images and presenting them in an environment that is essentially free of worry over diagnostic accuracy. Such exposure should only be beneficial in terms of building a comfort level among pathologists, especially as the technology continues to improve and new generations of pathologists encounter WSI technology throughout their residency training. The paper by Patterson et al, also includes a comprehensive list of survey questions that was pilot tested at a recent pathology informatics conference. The list of questions explores issues related to work environment in digital pathology as compared to light microscopy. I feel that this is an important and relatively underexplored aspect of digital pathology. It may be that pathologists will need to adjust their work environment so as to minimize the chance of being distracted by incoming e-mail or being interrupted by others while trying to sort out a live frozen section on their computer screen. As a pathologist who currently uses WSI to read frozen sections at my institution,[3] it has been my experience that I am significantly (dare I say without implying rigorous data collection with supporting statistics!) more likely to be interrupted when looking at cases on my office computer screen as opposed to using my microscope. While a lot of work remains to be done before pathology reaches the level of adoption seen in digital radiology, I agree with the comment by Patterson et al, the performance barriers are tractable. The big question is how long the process will take. Once the performance problems have been overcome, the full potential of WSI systems as a basis for diagnostic telepathology networks can be realized in terms of improving access to sub-specialty diagnostic opinions and providing pathology services to remote or underserviced locations.

The visual aids used in teaching and lecturing to students have clearly evolved over the last several decades. Illustrations on paper were replaced by carousels full of photographic slides, which in turn have been supplanted by Powerpoint™ presentations. At an arguably much slower pace, the method of teaching microscopic anatomy to students of the healthcare professions is also changing. For the majority of medical students graduating before 2000, the microscope and accompanying boxes of glass slides were the standard tools for learning both histology and pathology. Studying for exams also included reviewing kodachromes showing static fields-of-view at predetermined magnifications. Both of these methods of learning required the student to be physically present on campus. Pathology residents, when not learning at the microscope with the attending pathologist, must rely on teaching sets of glass slides that cannot leave the department. Group learning must either be done at a multiheaded microscope (which many times has fewer heads than people present) or by use of video technology attached to the microscope. With the advent of digital whole slide imaging (WSI) over the past several years, there is an opportunity to revolutionize the way teaching and learning are done for all students of medicine including doctors, nurses, medical technicians, histology technicians, cytology technicians, and others. Comparison of Digital Whole Slide Images with Glass Slides There are many advantages to using WSI when compared with traditional glass slides [Table 1]. Digital images can be standardized; all students will study the exact same tissue section. Sections on glass slides are inherently variable in quality and content.[1] Although admittedly subtle in some cases, these variabilities can be eliminated with digital imaging. The quality of the image can also be indefinitely maintained compared to glass slides that are vulnerable to fading, breaking, and vanishing.[2] One very helpful aspect of digital images is the use of a thumbnail image. As students are examining the image at higher magnification, they can always refer to the thumbnail for orientation.[3] This obviously is impossible with glass slides. Glass slides cannot be easily annotated with any precision, relying on relatively crude “dotting” for the purposes of highlighting a certain area of the slide. Digital images can have multiple annotations including arrows, circles, text, etc. placed exactly where needed. Portability is another benefit of using digital images. The use of microscopes obligates a student to remain on campus in order to study, review, etc. With WSI loaded onto a web-based server, study can occur wherever and whenever the student wishes.[2] WSI also makes simultaneous viewing of a particular field-of-view possible; a major advantage over glass slides. Viewing the image on a common computer monitor encourages discussion and collaboration between classmates.[1] Finally, storage of WSI becomes a matter of having enough server memory. As server space becomes less and less expensive, the cost and effort of storing and maintaining both the microscopes and glass slide sets will become comparatively more burdensome.[3] Some disadvantages that have become apparent with WSI include the dependence of the image quality on monitor resolution and the challenge of scanning tissue sections that have artifacts such as folds, etc.[3] Judicious choice of an acceptable non-cost prohibitive monitor is essential for an accurate image. As digital scanners become “smarter”, the flaws in a slide will likely become less of an issue. Table 1 Benefits of digital slide imaging

Background: Interest in developing more feasible and affordable applications of virtual microscopy in the field of cytology continues to grow. Aims: The aim of this study was to investigate the scanning parameters for the thyroid fine needle aspiration (FNA) cytology specimens. Subjects and Methods: A total of twelve glass slides from thyroid FNA cytology specimens were digitized at ×40 with 1 micron (μ) interval using seven focal plane (FP) levels (Group 1), five FP levels (Group 2), and three FP levels (Group 3) using iScan Coreo Au scanner (Ventana, AZ, USA) producing 36 virtual images (VI). With an average wash out period of 2 days, three participants diagnosed the preannotated cells of Groups 1, 2, and 3 using BioImagene's Image Viewer (version 3.1) (Ventana, Inc., Tucson, AZ, USA), and the corresponding 12 glass slides (Group 4) using conventional light microscopy. Results: All three raters correctly identified and showed complete agreement on the glass and VI for: 86% of the cases at FP Level 3, 83% of the cases at both the FP Levels 5 and 7. The intra-observer concordance between the glass slides and VI for all three raters was highest (97%) for Level 3 and glass, same (94%) for Level 5 and glass; and Level 7 and glass. The inter-rater reliability was found to be highest for the glass slides, and three FP levels (77%), followed by five FP levels (69.5%), and seven FP levels (69.1%). Conclusions: This pilot study found that among the three different FP levels, the VI digitized using three FP levels had slightly higher concordance, intra-observer concordance, and inter-rater reliability. Scanning additional levels above three FP levels did not improve concordance. We believe that there is no added benefit of acquiring five FP levels or more especially when considering the file size, and storage costs. Hence, this study reports that FP level three and 1 μ could be the potential scanning parameters for the thyroid FNA cytology specimens.

Recent advances in digital imaging is impacting the practice of pathology. One of the key enabling technologies that is leading the way towards this transformation is the use of whole slide imaging (WSI) which allows glass slides to be converted into large image files that can be shared, stored, and analyzed rapidly. Many applications around this novel technology have evolved in the last decade including education, research and clinical applications. This publication highlights a collection of abstracts, each corresponding to a talk given at Carnegie Mellon University's (CMU) Bioimaging Day 2014 co-sponsored by the Biomedical Engineering and Lane Center for Computational Biology Departments at CMU. Topics related specifically to digital pathology are presented in this collection of abstracts. These include topics related to digital workflow implementation, imaging and artifacts, storage demands, and automated image analysis algorithms.

Background: With the emerging role of digital imaging in pathology and the application of automated image-based algorithms to a number of quantitative tasks, there is a need to examine factors that may affect the reproducibility of results. These factors include the imaging properties of whole slide imaging (WSI) systems and their effect on the performance of quantitative tools. This manuscript examines inter-scanner and inter-algorithm variability in the assessment of the commonly used HER2/neu tissue-based biomarker for breast cancer with emphasis on the effect of algorithm training. Materials and Methods: A total of 241 regions of interest from 64 breast cancer tissue glass slides were scanned using three different whole-slide images and were analyzed using two different automated image analysis algorithms, one with preset parameters and another incorporating a procedure for objective parameter optimization. Ground truth from a panel of seven pathologists was available from a previous study. Agreement analysis was used to compare the resulting HER2/neu scores. Results: The results of our study showed that inter-scanner agreement in the assessment of HER2/neu for breast cancer in selected fields of view when analyzed with any of the two algorithms examined in this study was equal or better than the inter-observer agreement previously reported on the same set of data. Results also showed that discrepancies observed between algorithm results on data from different scanners were significantly reduced when the alternative algorithm that incorporated an objective re-training procedure was used, compared to the commercial algorithm with preset parameters. Conclusion: Our study supports the use of objective procedures for algorithm training to account for differences in image properties between WSI systems.

Several modes of telepathology exist including static (store-and-forward), dynamic (live video streaming or robotic microscopy), and hybrid technology involving whole slide imaging (WSI). Telepathology has been employed at the University of Pittsburgh Medical Center (UPMC) for over a decade at local, national, and international sites. All modes of telepathology have been successfully utilized to exploit our institutions subspecialty expertise and to compete for pathology services. This article discusses the experience garnered at UPMC with each of these teleconsultation methods. Static and WSI telepathology systems have been utilized for many years in transplant pathology using a private network and client-server architecture. Only minor clinically significant differences of opinion were documented. In hematopathology, the CellaVision(®) system is used to transmit, via email, static images of blood cells in peripheral blood smears for remote interpretation. While live video streaming has remained the mode of choice for providing immediate adequacy assessment of cytology specimens by telecytology, other methods such as robotic microscopy have been validated and shown to be effective. Robotic telepathology has been extensively used to remotely interpret intra-operative neuropathology consultations (frozen sections). Adoption of newer technology and increased pathologist experience has improved accuracy and deferral rates in teleneuropathology. A digital pathology consultation portal (https://pathconsult.upmc.com/) was recently created at our institution to facilitate digital pathology second opinion consults, especially for WSI. The success of this web-based tool is the ability to handle vendor agnostic, large image files of digitized slides, and ongoing user-friendly customization for clients and teleconsultants. It is evident that the practice of telepathology at our institution has evolved in concert with advances in technology and user experience. Early and continued adoption of telepathology has promoted additional digital pathology resources that are now being leveraged for other clinical, educational, and research purposes.

Pathology informatics encompasses digital imaging and related applications. Several specialized microscopy techniques have emerged which permit the acquisition of digital images ("optical biopsies") at high resolution. Coupled with fiber-optic and micro-optic components, some of these imaging techniques (e.g., optical coherence tomography) are now integrated with a wide range of imaging devices such as endoscopes, laparoscopes, catheters, and needles that enable imaging inside the body. These advanced imaging modalities have exciting diagnostic potential and introduce new opportunities in pathology. Therefore, it is important that pathology informaticists understand these advanced imaging techniques and the impact they have on pathology. This paper reviews several recently developed microscopic techniques, including diffraction-limited methods (e.g., confocal microscopy, 2-photon microscopy, 4Pi microscopy, and spatially modulated illumination microscopy) and subdiffraction techniques (e.g., photoactivated localization microscopy, stochastic optical reconstruction microscopy, and stimulated emission depletion microscopy). This article serves as a primer for pathology informaticists, highlighting the fundamentals and applications of advanced optical imaging techniques.

For many years pathologists have used Hematoxylin and Eosin (H&E), single marker immunohistochemistry (IHC) and in situ hybridization with manual analysis by microscopy or at best simple digital imaging. There is a growing trend to update pathology to a digital workflow to improve objectivity and productivity, as has been done in radiology. There is also a need for tissue-based multivariate biomarker assays to improve the accuracy of diagnostic, prognostic, and predictive testing. Multivariate tests are not compatible with the traditional single marker, manual analysis pathology methods but instead require a digital platform with brightfield and fluorescence imaging, quantitative image analysis, and informatics. Here we describe the use of the Hamamatsu NanoZoomer Digital Pathology slide scanner with HCImage software for combined brightfield and multiplexed fluorescence biomarker analysis and highlight its applications in biomarker research and pathology testing. This combined approach will be an important aid to pathologists in making critical diagnoses.