Whole-slide imaging in cytopathology: state of the art and future directions

Whole slide imaging (WSI) has been increasingly adopted for digital evaluation of surgical pathology specimens. Unlike histological slides, cytological preparations frequently display a heterogeneous distribution of cells throughout slides in different focal planes sometimes admixed with obscuring material, therefore requiring multiple scanning planes which significantly lengthens image acquisition and evaluation times. Although examination of digital images can be more advantageous than conventional glass slides, the challenges of focusing, scanning and screening cytological specimens and the associated increase in scan times and data storage needs have limited the routine application of WSI in cytopathology practice. Emerging digital systems designed to overcome image acquisition obstacles coupled with artificial intelligence algorithms augmenting screening of digital cytology slides offer innovative solutions to address these limitations. The aim of this review is to critically address the potential benefits and pitfalls of employing WSI in cytopathology practice and to introduce promising state-of-the-art solutions on the horizon.     

Utility of whole slide imaging and virtual microscopy in prostate pathology

Whole slide imaging (WSI) has been used in conjunction with virtual microscopy (VM) for training or proficiency testing purposes, multicentre research, remote frozen section diagnosis and to seek specialist second opinion in a number of organ systems. The feasibility of using WSI/VM for routine surgical pathology reporting has also been explored. In this review, we discuss the utility and limitations of WSI/VM technology in the histological assessment of specimens from the prostate. Features of WSI/VM that are particularly well suited to assessment of prostate pathology include the ability to examine images at different magnifications as well as to view histology and immunohistochemistry side-by-side on the screen. Use of WSI/VM would also solve the difficulty in obtaining multiple identical copies of small lesions in prostate biopsies for teaching and proficiency testing. It would also permit annotation of the virtual slides, and has been used in a study of inter-observer variation of Gleason grading to facilitate precise identification of the foci on which grading decisions had been based. However, the large number of sections examined from each set of prostate biopsies would greatly increase time required for scanning as well as the size of the digital file, and would also be an issue if digital archiving of prostate biopsies is contemplated. Z-scanning of glass slides, a process that increases scanning time and file size would be required to permit focusing a virtual slide up and down to assess subtle nuclear features such as nucleolar prominence. The common use of large blocks to process prostatectomy specimens would also be an issue, as few currently available scanners can scan such blocks. A major component of proficiency testing of prostate biopsy assessment involves screening of the cores to detect small atypical foci. However, screening virtual slides of wavy fragmented prostate cores using a computer mouse aided by an overview image is very different from screening glass slides using a microscope stage. Hence, it may be more appropriate in this setting to mark the lesional area and focus only on the interpretation component of competency testing. Other issues limiting the use of digital pathology in prostate pathology include the cost of high quality slide scanners for WSI and high resolution monitors for VM as well as the requirement for fast Internet connection as even a subtle delay in presentation of images on the screen may be very disturbing for a pathologist used to the rapid viewing of glass slides under a microscope. However, these problems are likely to be overcome by technological advances in the future.     

Use of whole slide imaging in surgical pathology quality assurance: design and pilot validation studies

By imaging large numbers of slides automatically at high resolution, modem automated whole slide imaging (WSI) systems have the potential to become useful tools in pathology practice. This article describes a pilot validation study for use of automated high-speed WSI systems for surgical pathology quality assurance (QA). This was a retrospective comparative study in which 24 full genitourinary cases (including 47 surgical parts and 391 slides) were independently reviewed with traditional microscopy and whole slide digital images. Approximately half the cases had neoplasia in the diagnostic line. At the end of the study, diagnostic discrepancies were evaluated by a pathology consensus committee. The study pathologists felt that the traditional and WSI methods were comparable for case review. They reported no difference in perceived case complexity or diagnostic confidence between the methods. There were 4 clinically insignificant discrepancies with the signed-out cases: 2 from glass slide and 2 with WSI review. Of the 2 discrepancies reported by the WSI method, the committee agreed with the reviewer once and the original report once. At the end of the study, the participants agreed that automated WSI is a viable potential modality for surgical pathology QA, especially in multifacility health systems that would like to establish interfacility QA. The participants felt that major issues limiting the implementation of WSI-based QA did not involve image acquisition or quality but rather image management issues such as the pathologist's interface, the hospital's network, and integration with the laboratory information system.     

Evaluation of whole slide image immunohistochemistry interpretation in challenging prostate needle biopsies

Whole slide images (WSIs), also known as virtual slides, can support electronic distribution of immunohistochemistry (IHC) stains to pathologists that rely on remote sites for these services. This may lead to improvement in turnaround times, reduction of courier costs, fewer errors in slide distribution, and automated image analyses. Although this approach is practiced de facto today in some large laboratories, there are no clinical validation studies on this approach. Our retrospective study evaluated the interpretation of IHC stains performed in difficult prostate biopsies using WSIs. The study included 30 foci with IHC stains identified by the original pathologist as both difficult and pivotal to the final diagnosis. WSIs were created from the glass slides using a scanning robot (T2, Aperio Technologies, Vista, CA). An evaluation form was designed to capture data in 2 phases: (1) interpretation of WSIs and (2) interpretation of glass slides. Data included stain interpretations, diagnoses, and other parameters such as time required to diagnose and image/slide quality. Data were also collected from an expert prostate pathologist, consensus meetings, and a poststudy focus group. WSI diagnostic validity (intraobserver pairwise kappa statistics) was "almost perfect" for 1 pathologist, "substantial" for 3 pathologists, and "moderate" for 1 pathologist. Diagnostic agreement between the final/consensus diagnoses of the group and those of the domain expert was "almost perfect" (kappa = 0.817). Except for one instance, WSI technology was not felt to be the cause of disagreements. These results are encouraging and compare favorably with other efforts to quantify diagnostic variability in surgical pathology. With thorough training, careful validation of specific applications, and regular postsignout review of glass IHC slides (eg, quality assurance review), WSI technology can be used for IHC stain interpretation.     

Evaluation of immunohistochemical staining using whole-slide imaging for HER2 scoring of breast cancer in comparison with real glass slides

Whole‐slide imaging (WSI) has been used for education and histological image preservation, and several studies have also reported its validity for practical pathological diagnosis. However, such studies employed materials stained with hematoxylin‐eosin (HE), and very few attempts have been made to use immunohistochemically stained materials for diagnostic purposes. In the present study, we investigated the availability of WSI diagnosis for immunohistochemically stained materials in place of routine glass slides. Thirty pathologists participated in a trial of HER2 expression diagnosis using WSI and compared the results with those obtained by light microscopy. The validity of WSI diagnosis (interobserver agreement) was rated as ‘substantial’ in comparison with glass slide diagnosis (κ‐value = 0.719). There was a tendency for observers to assign higher scores with WSI than with glass slides, probably because WSI requires slides to be scanned into a computer and observed via a monitor. Although we were able to demonstrate the potential utility of WSI for diagnosing immunostained materials, it must be borne in mind that there are some differences in visualization between WSI and glass slides.     

A validation study of whole slide imaging for primary diagnosis of lymphoma

Whole slide imaging (WSI) is being increasingly used worldwide. Although previous studies have asserted the validity of WSI diagnosis, they have primarily targeted only small specimens and excluded cases requiring immunohistochemistry or special staining, such as lymphoma. The purpose of this study was to evaluate the accuracy of WSI diagnosis of lymphoma, for which 240 biopsies and resections of lymphoma cases were selected from the study set of lymphomas. All slides including H&E, immunohistochemical and special staining were digitized using a WSI image scanner. An experienced pathologist performed the WSI diagnoses, which were compared with original diagnoses based on light microscopic examinations. Discrepancy between the two interpretations were classified into three categories: concordance, minor discrepancy (no clinical significance), and major discrepancy (with clinical significance). Overall concordance between the light microscopic and WSI diagnosis was found in 223 cases (92.92%; 95%CI = 88.90–95.82), minor discrepancy in fifteen (6.25%; 95%CI = 3.54–10.10), and major discrepancy in two (0.83%; 95%CI = 0.10–2.98). Diagnosis of lymphoma using WSI appeared to be mostly accurate, suggesting that WSI may be a reliable technology for the diagnosis of lymphoma.     

Whole-slide imaging: widening the scope of cytopathology

Whole slide imaging (WSI) is broadening the scope of cytopathology. Whole slide images are being used for telecytology, quality assurance activities (e.g. proficiency testing) and teaching (e.g. digital teaching sets and online virtual atlases). Progress in WSI technology that permits high resolution scanning, z-stacking, and hybrid robotic devices has encouraged the use of this imaging modality for cytology practice, education and research. However, widespread adoption in cytology still depends on overcoming barriers unrelated to cytology and challenges directly related to digitizing cytopathology slides. The aim of this article is to review WSI technology, applications and limitations specific to cytopathology.     

Digital breast pathology: validation and training for primary digital practice

Digital pathology is a technology which is transforming the way in which breast histopathology specimens are assessed, reported and reviewed. Large scale clinical laboratory deployments of whole slide imaging systems are occurring in diagnostic pathology departments across the world, requiring laboratory and diagnostic staff to navigate new skills and workflows. Transferring from conventional light microscopy assessment of breast specimens to the use of whole slide images (WSI) can be a challenging experience. This article describes an approach to training and validation for breast consultant histopathologists, which has been used and adapted at a number of sites. Examples of types of case that are suitable for training, and some of the potential “pitfalls” of digital reporting for the novice are described, and practical advice regarding clinical digital breast workflow is shared.     

Applications and implications of whole-slide imaging in breast pathology

Technological advances in whole slide imaging (WSI) technology and artificial intelligence (AI) applications in recent years have resulted in increasing adoption of this paradigm shift technology. This brings with it many advantages, new challenges, and potential adaptations to the microscopic assessment of specimens that pathologists need to be aware of. This article describes the applications and implications of WSI within the context of the reporting of breast pathology specimens. Challenging diagnostic entities in digital breast pathology are presented and the key areas in which AI could be useful in breast pathology are highlighted.     

Virtual microscopy and digital pathology in training and education

Traditionally, education and training in pathology has been delivered using textbooks, glass slides and conventional microscopy. Over the last two decades, the number of web-based pathology resources has expanded dramatically with centralized pathological resources being delivered to many students simultaneously. Recently, whole slide imaging technology allows glass slides to be scanned and viewed on a computer screen via dedicated software. This technology is referred to as virtual microscopy and has created enormous opportunities in pathological training and education. Students are able to learn key histopathological skills, e.g. to identify areas of diagnostic relevance from an entire slide, via a web-based computer environment. Students no longer need to be in the same room as the slides. New human-computer interfaces are also being developed using more natural touch technology to enhance the manipulation of digitized slides. Several major initiatives are also underway introducing online competency and diagnostic decision analysis using virtual microscopy and have important future roles in accreditation and recertification. Finally, researchers are investigating how pathological decision-making is achieved using virtual microscopy and modern eye-tracking devices. Virtual microscopy and digital pathology will continue to improve how pathology training and education is delivered.     

Whole-slide imaging: routine pathologic diagnosis

Digital pathology systems offer pathologists an alternate, emerging mechanism to manage and interpret information. They offer increasingly fast and scalable hardware platforms for slide scanning and software that facilitates remote viewing, slide conferencing, archiving, and image analysis. Deployed initially and validated largely within the research and biopharmaceutical industries, WSI is increasingly being implemented for direct patient care. Improvements in image quality, scan times, and imageviewing browsers will hopefully allow pathologists to more seamlessly convert to digital pathology, much like our radiology colleagues have done before us. However, WSI creates both opportunities and challenges. Although niche applications of WSI technology for clinical, educational, and research purposes are clearly successful, it is evident that several areas still require attention and careful consideration before more widespread clinical adoption of WSI takes place. These include regulatory issues, development of standards of practice and validation guidelines, workflow modifications, as well as defining situations where WSI technology will really improve practice in a cost-effective way. Current progress on these and other issues, along with improving technology, will no doubt pave the way for increased adoption over the next decade, allowing the pathology community as a whole to harness the true potential of WSI for patient care. The digital decade will likely redefine how pathology is practiced and the role of the pathologist.     

Whole slide imaging in cytopathology education

Rapid advances are occurring in the field of cytopathology education and training. Web-based cytopathology educational resources and whole slide imaging (WSI) have revolutionized cytopathology education and helped to centralize the cytopathology resources enabling simultaneous delivery of interactive cytopathology educational programs to a wide range of students and learners nationally and across the globe. WSI is playing a central role in digital pathology and are being utilized as an educational tool in many areas of pathology and cytopathology. This is mainly due to their easy access from anywhere and anytime, with no need for replication of glass slides or a big concern about the issue of protection of patient privacy. Today, WSI is used in a variety of educational settings, as a substitute to multi-headed microscopic sessions, multisite conferences, cytopathology web pages, self-assessment in cytology, cytology proficiency testing, virtual atlases, and very recently in scientific publications.     

Automated complete slide digitization: a medium for simultaneous viewing by multiple pathologists.

Developments in telepathology robotic systems have evolved the concept of a 'virtual microscope' handling 'digital slides'. Slide digitization is a method of archiving salient histological features in numerical (digital) form. The value and potential of this have begun to be recognized by several international centres. Automated complete slide digitization has application at all levels of clinical practice and will benefit undergraduate, postgraduate, and continuing education. Unfortunately, as the volume of potential data on a histological slide represents a significant problem in terms of digitization, storage, and subsequent manipulation, the reality of virtual microscopy to date has comprised limited views at inadequate resolution. This paper outlines a system refined in the authors' laboratory, which employs a combination of enhanced hardware, image capture, and processing techniques designed for telepathology. The system is able to scan an entire slide at high magnification and create a library of such slides that may exist on an internet server or be distributed on removable media (such as CD-ROM or DVD). A digital slide allows image data manipulation at a level not possible with conventional light microscopy. Combinations of multiple users, multiple magnifications, annotations, and addition of ancillary textual and visual data are now possible. This demonstrates that with increased sophistication, the applications of telepathology technology need not be confined to second opinion, but can be extended on a wider front.     

Validation of whole slide imaging for frozen section diagnosis of lymph node metastasis: A retrospective study from a tertiary care hospital in Thailand

BackgroundThe use of whole slide imaging (WSI) for frozen section (FS) diagnosis is helpful, particularly in the context of pathologist shortages. However, there is minimal data on such usage in resource-limited settings. This study aims to validate the use of WSI for FS diagnosis of lymph node metastasis using a low-cost virtual microscope scanner with consumer-grade laptops at a tertiary care hospital in Thailand.MethodsFS slides were retrieved for which the clinical query was to evaluate lymph node metastasis. They were digitized by a virtual microscope scanner (MoticEasyScan, Hong Kong) using up to 40× optical magnification. Three observers with different pathology experience levels diagnosed each slide, reviewing glass slides (GS) followed by digital slides (DS) after two weeks of a wash out period. WSI and GS diagnoses were compared. The time used for scanning and diagnosis of each slide was recorded.Results295 FS slides were retrieved and digitized. The first-time successful scanning rate was 93.6 %. The mean scanning time was 2 min per slide. Both intraobserver agreement and interobserver agreement of WSI and GS diagnoses were high (Cohen's K; kappa value >0.84). The time used for DS diagnosis decreased as the observer's experience with WSI increased.ConclusionsDespite varying pathological experiences, observers using WSI provided accurate FS diagnoses of lymph node metastasis. The time required for DS diagnoses decreased with additional observer's experience with WSI. Therefore, a WSI system containing low-cost scanners and consumer-grade laptops could be used for FS services in hospital laboratories lacking pathologists.     

An array microscope for ultrarapid virtual slide processing and telepathology. Design, fabrication, and validation study

This paper describes the design and fabrication of a novel array microscope for the first ultrarapid virtual slide processor (DMetrix DX-40 digital slide scanner). The array microscope optics consists of a stack of three 80-element 10 x 8-lenslet arrays, constituting a "lenslet array ensemble." The lenslet array ensemble is positioned over a glass slide. Uniquely shaped lenses in each of the lenslet arrays, arranged perpendicular to the glass slide constitute a single "miniaturized microscope." A high-pixel-density image sensor is attached to the top of the lenslet array ensemble. In operation, the lenslet array ensemble is transported by a motorized mechanism relative to the long axis of a glass slide. Each of the 80 miniaturized microscopes has a lateral field of view of 250 microns. The microscopes of each row of the array are offset from the microscopes in other rows. Scanning a glass slide with the array microscope produces seamless two-dimensional image data of the entire slide, that is, a virtual slide. The optical system has a numerical aperture of N.A.= 0.65, scans slides at a rate of 3 mm per second, and accrues up to 3,000 images per second from each of the 80 miniaturized microscopes. In the ultrarapid virtual slide processing cycle, the time for image acquisition takes 58 seconds for a 2.25 cm2 tissue section. An automatic slide loader enables the scanner to process up to 40 slides per hour without operator intervention. Slide scanning and image processing are done concurrently so that post-scan processing is eliminated. A virtual slide can be viewed over the Internet immediately after the scanning is complete. A validation study compared the diagnostic accuracy of pathologist case readers using array microscopy (with images viewed as virtual slides) and conventional light microscopy. Four senior pathologists diagnosed 30 breast surgical pathology cases each using both imaging modes, but on separate occasions. Of 120 case reads by array microscopy, there were 3 incorrect diagnoses, all of which were made on difficult cases with equivocal diagnoses by light microscopy. There was a strong correlation between array microscopy vs. "truth" diagnoses based on surgical pathology reports. The kappa statistic for the array microscopy vs. truth was 0.96, which is highly significant (z=10.33, p <0.001). There was no statistically significant difference between rates of agreement with truth between array microscopy and light microscopy (z=0.134, p >0.05). Array microscopy and light microscopy did not differ significantly with respect to the number/percent of correct decisions rendered (t=0.552, p=0.6376) or equivocal decisions rendered (t=2.449, p=0.0917). Pathologists rated 95.8% of array microscopy virtual slide images as good or excellent. None were rated as poor. The mean viewing time for a DMetrix virtual slide was 1.16 minutes. The DMetrix virtual slide processor has been found to reduce the virtual slide processing cycle more than 10 fold, as compared with other virtual slide systems reported to date. The virtual slide images are of high quality and suitable for diagnostic pathology, second opinions, expert opinions, clinical trials, education, and research.     

Reconciliation of diverse telepathology system designs. Historic issues and implications for emerging markets and new applications

Telepathology, the distant service component of digital pathology, is a growth industry. The word "telepathology" was introduced into the English Language in 1986. Initially, two different, competing imaging modalities were used for telepathology. These were dynamic (real time) robotic telepathology and static image (store-and-forward) telepathology. In 1989, a hybrid dynamic robotic/static image telepathology system was developed in Norway. This hybrid imaging system bundled these two primary pathology imaging modalities into a single multi-modality pathology imaging system. Similar hybrid systems were subsequently developed and marketed in other countries as well. It is noteworthy that hybrid dynamic robotic/static image telepathology systems provided the infrastructure for the first truly sustainable telepathology services. Since then, impressive progress has been made in developing another telepathology technology, so-called "virtual microscopy" telepathology (also called "whole slide image" telepathology or "WSI" telepathology). Over the past decade, WSI has appeared to be emerging as the preferred digital telepathology digital imaging modality. However, recently, there has been a re-emergence of interest in dynamic-robotic telepathology driven, in part, by concerns over the lack of a means for up-and-down focusing (i.e., Z-axis focusing) using early WSI processors. In 2010, the initial two U.S. patents for robotic telepathology (issued in 1993 and 1994) expired enabling many digital pathology equipment companies to incorporate dynamic-robotic telepathology modules into their WSI products for the first time. The dynamic-robotic telepathology module provided a solution to the up-and-down focusing issue. WSI and dynamic robotic telepathology are now, rapidly, being bundled into a new class of telepathology/digital pathology imaging system, the "WSI-enhanced dynamic robotic telepathology system". To date, six major WSI processor equipment companies have embraced the approach and developed WSI-enhanced dynamic-robotic digital telepathology systems, marketed under a variety of labels. Successful commercialization of such systems could help overcome the current resistance of some pathologists to incorporate digital pathology, and telepathology, into their routine and esoteric laboratory services. Also, WSI-enhanced dynamic robotic telepathology could be useful for providing general pathology and subspecialty pathology services to many of the world's underserved populations in the decades ahead. This could become an important enabler for the delivery of patient-centered healthcare in the future.     

International telecytology: Current applications and future potential

International telecytology can improve patient care by increasing access to regional and international expertise in cytopathology. The majority of international telecytology studies published to date have been based on static telepathology platforms. Overall concordance rates for these studies ranged from 71% to 93%. This is comparable to the concordance rates published for other studies comparing diagnoses made by digital still images to reference glass slides, which vary from 80% to 95%. Static telepathology systems are relatively cheap and easy to use, and have the potential to increase access to international experts in developing countries with limited resources. In contrast, resource‐rich academic and private medical centers can use whole slide digital imaging (WSI) for telecytology consultation, though few studies have been published addressing this topic. International telepathology consultation services with digital whole slide image capabilities have been established at several academic medical centers including the University of Pittsburgh Medical Center (UPMC) and the University of California at Los Angeles (UCLA), through the UCLA Center for Telepathology and Digital Pathology. In a small series of 20 telecytology cases submitted to UCLA from 2014 to 2017 (10 gynecologic and 10 fine needle aspiration cases), a meaningful diagnosis was rendered for 100% of cases, with 100% concordance between the submitting institution, versus consultation diagnosis provided by UCLA. These limited results are promising, and in the future both WSI and static telecytology consultation may have a place serving clinical needs in different practice settings.     

Loss of fidelity in scanned digital images compared to glass slides of brain tumors resected using cavitron ultrasonic surgical aspirator

Conversion of glass slides to digital images is necessary to capitalize on advances in computational pathology and could potentially transform our approach to primary diagnosis, research, and medical education. Most slide scanners have a limited maximum scannable area and utilize proprietary tissue detection algorithms to selectively scan regions that contain tissue, allowing for increased scanning speed and reduced file size compared to scanning the entire slide at high resolution. However, very small and faintly stained tissue fragments may not be recognized by these algorithms, leading to loss of fidelity in the digital image compared to the glass slides. Cavitron ultrasonic surgical aspirator (CUSA) is frequently used in brain tumor resections, resulting in highly fragmented specimens that are used for primary diagnosis. Here we evaluated the rate of loss of fidelity in 296 digital images from 40 CUSA‐resected brain tumors scanned using a Philips Ultra Fast Scanner. Overall, 54% of the slides (at least one from every case) showed loss of fidelity, with at least one tissue fragment not scanned at high resolution. The majority of the missed tissue fragments were small (<0.5 mm), but rare slides were missing fragments greater than 5 mm in greatest dimension. In addition, 19% of the slides with missing tissue showed no indication of loss of fidelity in the digital image itself; the missing tissue could only be appreciated upon review of the glass slides. These results highlight a potential liability in the use of digital images for primary diagnosis in CUSA‐resected brain tumor specimens.