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.     

Progression in diagnostic pathology; development of virtual microscopy and its applications

Many systems have already been designed and successfully used for clinical laboratory and pathological examination. The evolution of image analysis was enabled when analog images of the original glass slides could be transferred to digital images with the rapid development of virtual microscopy and virtual slides depended upon computer technologies. Today, whole slide can be acquired by virtual microscopes. The applications of virtual microscopy and virtual slides for teaching, diagnosis, telepathology, and research are more widely used than those of real microscope and real glass slides. In traditional cancer diagnosis, pathologists examine biopsies to make diagnostic assessments largely based on two-dimensional cell morphology and tissue distribution. These assessments are subjective and often show considerable variability. However, automated cancer diagnostic system based on three-dimensional image analysis based on nuclear bulging sign enables objective judgments using quantitative measurements. We expect that the shortage of pathologists will be improved when an automated cancer diagnosis system is developed.     

Values and limits in fine needle aspiration in the diagnosis of Warthin tumour of the parotid gland

Recent paper state that cytological diagnosis of Warthin Tumour of the parotid gland is poorly reproducible and that aspiration procedures damage the tumoral tissue as to make difficult the histological diagnosis. Aim of the present study is to review cytological sampling performed with fine needle sampling without aspiration and to compare them with the relative surgical specimens in order to determine the value of fine needle aspiration in the diagnosis of Warthin Tumour and to quantify' the damage in histological tissue. MATERIALS AND METHOD: All cases with cytological and/or histological diagnosis of Warthin Tumour during the period 1/1/98 to 30/6/04 were retrieved. In all cases both cytological and histological slides were reviewed and compared. In all cases fine needle aspiration (FNA) was performed according to the technique described by Zajdela et al. in 1987. A cytologic sample was considered diagnostic when there were in the same sample mucoid substance, lymphocytes scattered with a range of maturation and oncocytes organized in flat sheets of cells. RESULTS: 38 cases were selected corresponding to 37 patients (1 case of Warthin Tumour bilateral): all tumors were localized in the parotid gland. Fine needle sampling were 43. Nine cytological sample (20.9%) were inadequate. Sensitivity and specificity, in the preent series, had the same value, being 97.1%. A spectrum of histologic alterations were observed in 30/38 cases (79%). These alterations were squamous metaplasia (7 cases), acute and chronic hemorrhage and inflammation with multinucleated giant cells (26 cases) and granulation tissue with subsequent fibrosis (24 cases). In all cases the histologic alterations from fine needle were focal, limited to less than 20% of the tumoral area (first category of Batsakis 'classification), and did not prevent the histologic diagnosis of Warthin Tumour. CONCLUSIONS: These data suggest that fine needle aspiration without aspiration is a valuable tool for the per-operative diagnosis of Warthin Tumour. In addition it creates only minor histological changes, that always allow the histological diagnosis.     

The diagnostic value of the thinprep pap test in endometrial carcinoma: A prospective study with histological follow‐up

Case‐control studies have demonstrated that the ThinPrep Pap test may provide improved detection of endometrial carcinoma. The purpose of this study is to prospectively examine the diagnostic potential of the ThinPrep Pap test in the detection of endometrial carcinoma. ThinPrep Pap test slides were collected from high‐risk patient groups. Pap‐stained slides were reviewed and the cytological diagnosis was rendered independently by investigators. Each case was assigned to one of the four diagnostic categories: within normal limit (WNL); atypical glandular cells (AGC); atypical endometrial cells (AEC); or adenocarcinoma, probably endometrial origin. After cytological diagnosis was made, the histological follow‐up diagnosis was obtained through the laboratory information system and the cyto‐histological correlation was analyzed. Of 106 patients identified, 60 had histological follow‐up. For all eight cases interpreted by cytology as positive, endometrial carcinoma was confirmed histologically. Among 25 patients with normal endometrial cells present, histological follow‐up showed benign endometrium. Among 17 cases interpreted cytologically as AEC, 14 cases (82.4%) had benign histological follow‐up and 3 cases (17.6%) had endometrial carcinoma. All 11 cases (100%) classified as AGC had benign histological follow‐up. The sensitivity and specificity of detecting endometrial malignancy were 72.7% and 100%, respectively. The positive predictive value was 100%. In this prospective study, we demonstrated that the Thin Prep Pap test had a reasonably high sensitivity and/or specificity in detecting endometrial carcinoma. Diagn. Cytopathol. 2013. © 2012 Wiley Periodicals, Inc.     

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.     

Virtual microscopy: high resolution digital photomicrography as a tool for light microscopy simulation

Recent advances in microcomputers and high resolution digital video cameras provide pathologists the opportunity to combine precision optics with digital imaging technology and develop new educational and research tools. We review recent advances in virtual microscopy and describe techniques for viewing digital images using a microcomputer-based workstation to simulate light microscopic examination, including scanning at low power to select features of interest and zooming to increase magnification. Hardware and software components necessary to acquire digital images of histological and cytological slides, and closely simulate their examination under a light microscope are discussed. The workstation is composed of a MicroLumina digital scanning camera (Leaf Systems, Southborough, MA), light microscope (Olympus Optical Co., Lake Success, NY), Pentium (Intel Corp., Santa Clara, CA) 166 MHz microcomputer configured with 64 megabytes of random access memory (RAM), a MGA Millenium Powerdesk graphics card (Matrox Graphics, Inc., Montreal, Canada) and Photoshop software (Adobe Systems Inc., San Jose, CA) running in a Windows 95 (Microsoft Corp., Redmond, WA) environment. Images with spatial resolutions of up to 2700 x 3400 pixels in 36-bit color, can be displayed simultaneously as distinct images in a montage, or merged into a single composite image file to highlight significant features of a histological or cytological slide. These image files are saved in Joint Photographers Experts Group (JPEG) format using compression ratios of up to 80:1 without detectable visual degradation. The advantages and technical limitations of various workstation components are addressed and applications of this technology for pathology education, proficiency testing, telepathology, and database development are discussed.     

French pathologist' implication in cervical cancer screening

The cytological test issue mostly from individual opportunistic or for some department from organized screening programme, must benefit of the same quality in all the procedures. Pathologists are very implicated in. The purpose of this document is to summarize cytopathologists' initiatives and to present their specific tools for that. The French association for quality assurance in pathology (AFAQAP) has been validated by HAS in March 2006 for EPP. Commission 1 of AFAQAP organized an annual voluntary test for diagnostic evaluation since 2000 with the adoption of new technologies (CD-rom, virtual digital slides), and in 2005 a test of Pap Smear reporting quality concerning TBS 2001 use. A referring evaluation on this theme is in press. Furthermore pathologists made tools are available to codify lesions they diagnose (ADICAP), to collect and to study data (FCRISAP), to standardize the histological and cytological reports (CRFS), to evaluate the procedures and structural requirements and techniques. Pathologists participate in the elaboration of national guidelines (ANAES 1998 et 2002, DGS 2006) and performance indicators with InVS. The cervix cancer screening programme must take in account and valorises the role of pathologists according to assessment of efficacy and evaluation of the screening.     

Implementation of virtual microscope slides in the annual pathobiology of cancer workshop laboratory

Virtual slides are digital facsimiles of glass microscope slides that, when viewed with a pan and zoom viewer, can emulate viewing a glass slide with a traditional microscope. Based on successful implementation of virtual slides in medical student histology and pathology courses at the University of Iowa, we developed a plan to evaluate the use of virtual slides in the American Association for Cancer Research's annual Pathobiology of Cancer Workshop. In this Workshop, nonphysician predoctoral students and postdoctoral fellows working in cancer research explore the morphological, clinical, and molecular aspects of human cancer. Over the course of a week, students examine approximately 100 glass slides in microscope laboratories, facilitated by senior cancer investigators. The goal of the present study was to evaluate virtual slides as a teaching modality in these laboratories, not as a replacement for traditional microscopy, but rather in terms of their utility in facilitating student learning as they examine glass slides with a traditional microscope. Evaluation by questionnaire indicated that virtual slides enhanced students' ability to grasp morphological features better than the traditional photomicrographs. The results of this implementation suggest that virtual slide technology may be successfully extended to other educational venues where traditional microscopy and photomicrographs are currently used.     

Use of imprint cytology for assessment of surgical margins in lumpectomy specimens of breast cancer patients

In the past several years, breast-conservation therapy has provided an alternative to mastectomy. In order to reduce the subsequent local tumor recurrence, it is critical that all the measures are in place to find the residual foci of occult microscopic tumor at the time of the initial lumpectomy procedure. An accepted method to evaluate the lumpectomy margins for presence of residual tumor is the use of imprint cytology (also called touch-prep), which is assessment of the presence or absence of the tumor cells by cytological preparation. This is a rapid, cost effective, and easy to use procedure with added advantage of saving tissue for permanent sectioning and rendering a definitive diagnosis. In this report, we present our experience using intraoperative imprint cytology for evaluation of the status of lumpectomy specimens in breast cancer patients. The objective of this study was to evaluate the diagnostic accuracy of intraoperative imprint cytology for assessment of surgical resection margins in lumpectomy margins of patients with breast carcinoma. This is a retrospective study of 100 cases of breast lumpectomy specimens, which had undergone intraoperative imprint cytology. The cases were retrieved from the archived files of the University of Florida, Department of Pathology at Shands Jacksonville. The results of intraoperative imprint cytology were compared with the histological findings of the corresponding permanent sections of the same cases as the gold standard. Overall, we reviewed 510 cytology imprint slides, which were obtained from 100 lumpectomy specimens. Among these cases, 37 slides from 22 cases were reported positive and the remaining were negative. Only eight slides from six cases of lumpectomy showed discrepancy between the result of intraoperative imprint cytology and the permanent sections of the same cases. In our study, intraoperative imprint cytology showed a sensitivity of 97%, specificity of 99%, with positive predictive value of 84%, and negative predictive value of 99%. This study demonstrates that intraoperative imprint cytology can be used as a reliable diagnostic procedure for the evaluation of the status of lumpectomy margins in breast cancer patients.     

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.     

Virtual laboratory manual for microscopic anatomy

Using digital technology, we have assembled a virtual laboratory manual (VLM) that is a Web-based copy of our traditional laboratory manual. The VLM is used to enhance traditional laboratory instruction in histology. For each reference in the VLM to either a histological slide or an electron micrograph (EM), hyperlinks are included that download digital images derived from the students' glass slide sets or scanned EMs. The VLM serves as an atlas of digital images for concurrent study of similar sections by light microscopy during laboratory sessions. In addition, students can review the images from the VLM at remote locations. We have encouraged continued use of light microscopes in laboratories by basing the majority of practical examination identifications on analysis of marked histological slides that require students to use their own microscopes. The VLM provides the convenience of a Web-based resource with high-quality images, yet allows retention of the many excellent traditional aspects of our course. An example of a VLM laboratory on epithelium is available online (http://users.von.uc.edu/michaeje/VLM-Epithelium/exLab4.pdf).     

Intraoperative examination in neuropathology: proceedings evaluations

INTRODUCTION: In the neuropathological intraoperative diagnostic is extremely important having guide lines to obtain a lot of information about the slide. In our study we have considered only the intraoperative the cases diagnosed within a month comparing the histological evaluation using the cryostat and the cytological examination of touch or smear preparation. MATERIALS AND METHODS: Our study includes 7 meningiomas, 2 metastatic carcinomas, 1 anaplastic astrocitoma, 1 mieloplaxis tumor, 1 anaplastic chordoma. For the cytological examination we have got ready 8 touch or smear preparations and we have executed 4 proceedings: stainings with Giemsa, toluidine blue and haematoxylin and eosin, after fixation with 95% alcohol and staining with haematoxylin and eosin after fixation with alcohol and a small quantity of acetic acid. For the histological examination we have got ready 8 preparations, using the cryostat. We have fixed the slides with 95% alcohol or 10% formalin and stained them with haematoxylin and eosin. In the 50% of the cases we have treated the sections with microwave at 400W. RESULTS: Two pathologists have examinated the 192 sections prepared, judging each slide considering the legibility: unsatisfactory, quite good, good and excellent. CONCLUSIONS: Considering the cytological examination, Giemsa and toluidine blue permit the best legibility of nuclear and cytoplasmatic morphological details. Legibility is worse in staining with haematoxylin and eosin. Addition of acetic acid makes variable results. In the histological slides the best result are obtained using 95% alcohol for fixing. Microwave use doesn't determine improvement.     

Virtual microscopy for learning and assessment in pathology

Virtual slides are high-magnification digital images of tissue sections, stored in a multi-resolution file format. Using appropriate software, these slides can be viewed in a web browser in a manner that closely simulates examination of glass slides with a real microscope. We describe the successful implementation of teaching microscopic pathology with virtual slides and, for the first time, their use in summative assessment. Both students and teaching staff readily adapted to the use of virtual microscopy. Questionnaire feedback from students strongly indicated that virtual slides solved a number of problems in their learning, while providing good to excellent image quality. A deliberate policy of allocating two students per workstation promoted collaboration and helped to maintain interest in microscopic pathology. The use of a secure browser facilitated assessment using virtual slides, with no technical or security issues arising despite high peak demand. The new Medicine programme at the University of New South Wales will exclusively utilize virtual microscopy for the study of both histology and histopathology. We believe that the use of high-quality learning resources such as virtual slides can ensure that microscopic examination of tissues remains both meaningful and interesting.     

Virtual microscopy in pathology teaching and postgraduate training (continuing education)

As with conventional microscopy, virtual microscopy permits histological tissue sections to be viewed on a computer screen with a free choice of viewing areas and a wide range of magnifications. This, combined with the possibility of linking virtual microscopy to E-Learning courses, make virtual microscopy an ideal tool for teaching and postgraduate training in pathology. Uses of virtual microscopy in pathology teaching include blended learning with the presentation of digital teaching slides in the internet parallel to presentation in the histology lab, extending student access to histology slides beyond the lab. Other uses are student self-learning in the Internet, as well as the presentation of virtual slides in the classroom with or without replacing real microscopes. Successful integration of virtual microscopy depends on its embedding in the virtual classroom and the creation of interactive E-learning content. Applications derived from this include the use of virtual microscopy in video clips, podcasts, SCORM modules and the presentation of virtual microscopy using interactive whiteboards in the classroom.     

Combined cytological and histological diagnosis of hepatocellular carcinoma in ultrasonically guided fine needle biopsy specimens

This study evaluates the usefulness of a combined cytological and histological approach to the diagnosis of hepatocellular carcinoma (HCC) when applied to fine needle biopsy specimens obtained under ultrasonic guidance. The material, aspirated from 51 focal liver lesions, was handled in such a way that there was sufficient material for both cytological and histological (cell block) assessment. Of the 29 cases of HCC studied, a confident cytological diagnosis was made in 23 (79%). In the remaining six cases, the cytological features were considered to be suspicious but not diagnostic of HCC. Examination of cell blocks in the six cases enabled a confident diagnosis of HCC to be made in all cases. This was due to the supplementary visual information provided by the histological features, particularly the pattern of arrangement of the tumour cells.     

Factors to keep in mind when introducing virtual microscopy

Digitization of glass slides and delivery of so-called virtual slides (VS) emulating a real microscope over the Internet have become reality due to recent improvements in technology. We have implemented a virtual microscope for instruction of medical students and for continuing medical education. Up to 30,000 images per slide are captured using a microscope with an automated stage. The images are post-processed and then served by a plain hypertext transfer protocol (http)-server. A virtual slide client (vMic) based on Macromedia's Flash MX, a highly accepted technology available on every modern Web browser, has been developed. All necessary virtual slide parameters are stored in an XML file together with the image. Evaluation of the courses by questionnaire indicated that most students and many but not all pathologists regard virtual slides as an adequate replacement for traditional slides. All our virtual slides are publicly accessible over the World Wide Web (WWW) at . Recently, several commercially available virtual slide acquisition systems (VSAS) have been developed that use various technologies to acquire and distribute virtual slides. These systems differ in speed, image quality, compatibility, viewer functionalities and price. This paper gives an overview of the factors to keep in mind when introducing virtual microscopy.Universal resource locators (URLs) visited at the time of writing (June 2005) may change or vanish with lapse of time. Therefore and because of usability, we have created a web page with all URLs that will be updated on a regular basis: .     

Virtual microscopy: an evaluation of its validity and diagnostic performance in routine histologic diagnosis of skin tumors

Digitization of histologic slides is associated with many advantages, and its use in routine diagnosis holds great promise. Nevertheless, few articles evaluate virtual microscopy in routine settings. This study is an evaluation of the validity and diagnostic performance of virtual microscopy in routine histologic diagnosis of skin tumors. Our aim is to investigate whether conventional microscopy of skin tumors can be replaced by virtual microscopy. Ninety-six skin tumors and skin-tumor–like changes were consecutively gathered over a 1-week period. Specimens were routinely processed, and digital slides were captured on Mirax Scan (Carl Zeiss MicroImaging, Göttingen, Germany). Four pathologists evaluated the 96 virtual slides and the associated 96 conventional slides twice with intermediate time intervals of at least 3 weeks. Virtual slides that caused difficulties were reevaluated to identify possible reasons for this. The accuracy was 89.2% for virtual microscopy and 92.7% for conventional microscopy. All κ coefficients expressed very good intra- and interobserver agreement. The sensitivities were 85.7% (78.0%-91.0%) and 92.0% (85.5%-95.7%) for virtual and conventional microscopy, respectively. The difference between the sensitivities was 6.3% (0.8%-12.6%). The subsequent reevaluation showed that virtual slides were as useful as conventional slides when rendering a diagnosis. Differences seen are presumed to be due to the pathologists' lack of experience using the virtual microscope. We conclude that it is feasible to make histologic diagnosis on the skin tumor types represented in this study using virtual microscopy after pathologists have completed a period of training. Larger studies should be conducted to verify whether virtual microscopy can replace conventional microscopy in routine practice.     

Teaching medical histology at the University of South Carolina School of Medicine: Transition to virtual slides and virtual microscopes

We describe how the histology course we teach to first-year medical students changed successfully from using glass slides and microscopes to using virtual slides and virtual microscopes. In 1988, we taught a classic medical histology course. Subsequently, students were loaned static labeled images on projection slides to introduce them to their microscope glass slides, and we made laser disks of histological images available in the teaching lab. In 2000, we placed the static labeled images and laboratory manual on the Web. We abandoned the Web-based approach in 2001. Faculty selected specific areas on microscope glass slides in student collections for scanning at a total magnification of 40, 100, 200, or 400. Christopher M. Prince of Petro Image, LLC, scanned the glass slides; digitized, encoded, and compressed (95%) the images; and placed them on CD-ROMs. The scanned images were viewed up to a magnification of 400 using the MrSID viewer (LizardTech software) and the computer as a virtual microscope. This viewer has many useful features, including effective microscope and telescope functions that provide greater versatility for sample study and speed in localizing structures than was possible with the actual microscope. Image detail is indistinguishable from that viewed under the light microscope at equivalent magnifications. Static labeled images were also placed on CD-ROMs to introduce students to the virtual slides. Students could view all the images on their CD-ROMs at any time and in any place with their laptop computers without going online. Students no longer rented light microscopes in 2002. Both students and faculty have shown strong support for using this approach to teaching histology during the past 2 years.