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.     

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.     

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.     

How many lymph nodes to stage colorectal carcinoma?

This study aimed to establish the number of cassettes that should be filled with lymph nodes to stage a colorectal carcinoma as Dukes's stage C. The records from the Oxford Colorectal Cancer database of all patients diagnosed with Dukes's stage C cancer from late 1988 to early 1993 were reviewed. Each slide of lymph nodes was examined to determine how many slides needed to be looked at to find the first positive lymph node. The resected specimens were not fat cleared but dissected manually in a routine fashion. One hundred and eight slides were retrieved. The mean total lymph node harvest was 8.44 for each patient. Ninety eight patients (90.7%) had positive lymph nodes on the first slide with an average of 3.42 lymph nodes on each slide, of which a mean of 1.82 were positive. For nine patients, two slides were required to make a diagnosis of lymph node involvement, and for one patient the first three slides needed inspection to establish Dukes's stage C. In conclusion, using a routine technique to obtain lymph nodes from colorectal cancer specimens 99% of Dukes's stage C colorectal carcinoma can be found by filling two cassettes with lymph nodes.     

Development of a database system and image viewer to assist in the correlation of histopathologic features and digital image analysis with clinical and molecular genetic information

Pathologists are required to integrate data from multiple sources when making a diagnosis. Furthermore, whole slide imaging (WSI) and next generation sequencing will escalate data size and complexity. Development of well‐designed databases that can allow efficient navigation between multiple data types is necessary for both clinical and research purposes. We developed and evaluated an interactive, web‐based database that integrates clinical, histologic, immunohistochemical and genetic information to aid in pathologic diagnosis and interpretation with nine lung adenocarcinoma cases. To minimize sectioning artifacts, representative blocks were serially sectioned using automated tissue sectioning (Kurabo Industries, Osaka Japan) and selected slides were stained by multiple techniques, (hematoxylin and eosin [H&E], immunohistochemistry [IHC] or fluorescence in situ hybridization [FISH]). Slides were digitized by WSI scanners. An interactive relational database was designed based on a list of proposed fields covering a variety of clinical, pathologic and molecular parameters. By focusing on the three main tasks of 1.) efficient management of textual information, 2.) effective viewing of all varieties of stained whole slide images (WSI), and 3.) assistance in evaluating WSI with computer‐aided diagnosis, this database prototype shows great promise for multi‐modality research and diagnosis.     

The spectrum of pathological diagnoses in non-sentinel axillary lymph node biopsy: A single institution's experience

Although axillary lymphadenopathy is a common clinical encounter, systemic evaluation of non-sentinel lymph node biopsy is sparse. We reviewed our institution's 15-year experience to delineate the spectrum of diagnoses in non-sentinel axillary lymph nodes. 1165 non-sentinel axillary lymph node biopsies were retrieved and the diagnosis and relevant clinical information was reviewed. This spectrum of diagnoses was further stratified by gender, age, and oncologic history. The spectrum of diagnoses included: breast carcinoma (27.6%), lymphoma (29.2%), melanoma (3.5%), other carcinoma (2.9%), sarcoma (0.4%), and benign changes (36.3%). The most common diagnoses in men were lymphoma (61.8%) and benign changes (23.6%); while in women they were benign change (41.2%), breast carcinoma (37.8%) and lymphoma (16.7%). Besides benign changes, lymphoma and breast carcinoma were most common in women younger and older than 30 years, respectively. In patients with a history of malignancy, the most common diagnoses were metastasis from the known tumor and benign change; while in patients with a negative oncologic history and female patients without a history of breast cancer, the diagnosis was generally either lymphoma or benign change. Anaplastic large cell lymphoma was rare but may be mistaken as metastatic carcinoma thus a high index of suspicion is warranted. Thus through retrospective review of a large cohort of non-sentinel axillary lymph node biopsies, we described the spectrum of pathological entities based on the gender, age, and clinical history, which could provide valuable information for further work-up of axillary lymph node biopsy.     

Integration of digital pathology in multidisciplinary breast site group rounds

Whole slide scanning and digitizing an entire glass slide technology opens multiple opportunities for integration in clinical practice. Clinical applications other than primary diagnosis include the use of digitized slides in multidisciplinary rounds. Integration of this emerging technology requires not only adaptation by pathologists but also investment in infrastructure for hardware and software components, electronic storage solutions, support from clinicians and hospital administration as well as training personal. The process of replacing conventional glass with digitized slides in pathology case presentation in multidisciplinary rounds is discussed highlighting the strengths and weaknesses of this transition. Successful implementation relies heavily on careful preliminary workflow process design and support from leaders within Anatomic Pathology and the cancer center.     

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.     

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.     

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.     

Effect of Display Resolution on Time to Diagnosis with Virtual Pathology Slides in a Systematic Search Task

Performing diagnoses using virtual slides can take pathologists significantly longer than with glass slides, presenting a significant barrier to the use of virtual slides in routine practice. Given the benefits in pathology workflow efficiency and safety that virtual slides promise, it is important to understand reasons for this difference and identify opportunities for improvement. The effect of display resolution on time to diagnosis with virtual slides has not previously been explored. The aim of this study was to assess the effect of display resolution on time to diagnosis with virtual slides. Nine pathologists participated in a counterbalanced crossover study, viewing axillary lymph node slides on a microscope, a 23-in 2.3-megapixel single-screen display and a three-screen 11-megapixel display consisting of three 27-in displays. Time to diagnosis and time to first target were faster on the microscope than on the single and three-screen displays. There was no significant difference between the microscope and the three-screen display in time to first target, while the time taken on the single-screen display was significantly higher than that on the microscope. The results suggest that a digital pathology workstation with an increased number of pixels may make it easier to identify where cancer is located in the initial slide overview, enabling quick location of diagnostically relevant regions of interest. However, when a comprehensive, detailed search of a slide has to be made, increased resolution may not offer any additional benefit.     

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.     

Usefulness of Ultrafast Papanicolaou-stained scrape preparations in intraoperative management of thyroid lesions.

The value of intraoperative pathologic consultation (including frozen-section [FS]analysis) in surgical management of thyroid lesions has decreased since the advent of preoperative fine-needle aspiration. This study presents an intraoperative consultation experience with tumor scrape preparations stained with Ultrafast Papanicolaou stain and FS analysis in 93 cases of thyroid nodules and 4 lymph nodes (as part of lymph node dissection due to thyroid malignancy) in 87 patients at the University of Pisa, Pisa, Italy. FS was performed in 41 cases (48%). The scrape preparation diagnoses included 71 malignancies (69 papillary and 2 poorly differentiated carcinomas); 1 "suspicious for carcinoma"; 9 follicular neoplasms; 13 benign (9 "no tumor seen," 1 thyroiditis, 1 hyperplastic nodule, and 2 nodular goiters); and 3 nondiagnostic cases. The final diagnosis agreed with the scrape diagnosis in 95 cases (98%). FS agreed with the final diagnosis in 29 cases (71%). We suggest that intraoperative scrape preparations of thyroid nodules stained with the Ultrafast Papanicolaou method might be a useful adjunct in the intraoperative management of thyroid nodules.     

A study of intraoperative rapid frozen section diagnosis focusing on accuracy and quality assessment]

Accuracy of frozen section (FS) diagnoses was investigated in a consecutive series of 1129 intraoperative consultations. In our series, the correct diagnosis including clinically not serious minor errors was made in 96.9% of the cases. Whereas the accuracy ratio of FS diagnosis for unknown pathologic process decreased to 92.2%. Among all the types of tissues, central nervous system was shown to be the most frequently handled for FS, followed by lung/bronchus, breast, liver/biliary tract, lymph node and so forth. The requesting ratio of FS, on the other hand, tended to be high in the following specialties; neurosurgery (46.5%), thoracic surgery (19.0%), general surgery, (10.0%). Deferred diagnosis with a provisional diagnosis and misinterpretations in histology typing without serious clinical problem accounted for 2.7% and 3.1% respectively. Causes of erroneous diagnoses seemed to be multifactorial, such as inappropriate sampling, diagnosis on poor quality histology sections, lack of clinical information, lack of enough experience in FS practice of pathologists, or a combination of more than two of them, though inevitable cases showing minimal cytological and structural atypia were included. Intraoperative consultation by FS diagnosis is now essential to serve a good quality medication to patients. It is thus necessary that to establish a tight peer review system and also to provide an education program with regard to practice in FS diagnosis particularly for young pathologists.     

Telecytology: Intraobserver and interobserver reproducibility in the diagnosis of cervical-vaginal smears

Telecytologic diagnosis of cervical-vaginal smears is potentially useful because it could allow more efficient use of cytopathologist resources and expertise. A pathologist in one location could, in principle, review cytotechnologists' findings using a video display hundreds or thousands of miles away. Currently, bandwidth restrictions limit practical implementation of such a system to review of fields that had been selected for review by the cytotechnologist. The purpose of our investigation was to evaluate how well this type of review correlates with a review in which the entire slide is available for examination by the pathologist. We prospectively selected 100 consecutive cervical-vaginal smears over an 11-day period in August 1999. For each smear, 4 to 12 fields containing abnormal cells from each slide were digitally imaged. Each of 3 pathologists reviewed all digitized images and all glass slides. Diagnoses based on selected digitized images were compared with those based on conventional pathologist review. The kappa statistic, a measure of chance-corrected agreement (reproducibility), was calculated in each setting. Overall, intraobserver and interobserver reproducibility of cervical-vaginal smear diagnoses is fair to excellent. The use of remote digital images for pathologist review did not introduce large (2-step) diagnostic disagreements. The disagreement between a pathologist's glass slide and digital diagnoses is less than that for different pathologists reviewing glass slides, although interobserver differences were even greater in the interpretation of digital images.     

Cellular dyscohesion in fine-needle aspiration of breast carcinoma. Prognostic indicator for axillary lymph node metastases?

The role of fine-needle aspiration (FNA) in the diagnosis of breast carcinoma is established. We evaluated whether the degree of cellular dyscohesion and the nuclear grade in FNA material of breast carcinomas are reliable prognostic predictors for ipsilateral axillary lymph node metastasis. FNA specimens from 98 women with infiltrating ductal and infiltrating lobular carcinomas were evaluated by 2 observers for degree of cellular dyscohesion and nuclear grade. Follow-up specimens from lumpectomy and/or mastectomy with axillary dissection were available for each patient. By univariate analysis, degree of cellular dyscohesion and nuclear grade were not predictive of axillary lymph node metastasis regardless of tumor size. High histologic grade, size greater than 2 cm, and patient age younger than 52 years were significant predictors of metastasis. By multivariate analysis, size greater than 2 cm and age younger than 52 years were statistically significant for lymph node metastasis. In contrast with a published study, the results of the present study fail to show cellular dyscohesion in FNA specimens as predictive of lymph node metastasis; however, the scoring method for determining the degree of cellular dyscohesion is reproducible between 2 independent observers.     

Z‐axis video for cytology database is a useful tool for the case presentation prior to the cytology training workshop

We have built a database on the Internet managing z‐axis video for cytology (Zavic), and report on a new style of case discussion supported by the Zavic database. Z‐axis video for cytology is a movie file derived from the video recording of a microscopic field with changes in the focus. We used it for the case presentation of EUS‐FNA of pancreatic lesions on the Internet prior to a training workshop. The attendees were asked to observe the Zavic and to make diagnoses of 20 cases before the workshop. Fourteen attendees also observed lesions under a microscope on that day, and the results were compared with those of Zavic observation. The evaluation of the Zavic database (DB) was surveyed by a questionnaire. The average number of accurate diagnoses by 46 Zavic observers was 10.8. These accuracies for those who observed both the Zavic and glass slides were 11.57 and 11.43, respectively, for the videos and slides. Compared with Zavic observation alone, the diagnoses with glass slide observation were shifted to a correct diagnosis in two cases, but were shifted to an incorrect diagnosis in two cases. Approximately 60% of Zavic observers replied in the questionnaire that the movies on Zavic DB started to play within 3–4 seconds after clicking the play button. We successfully carried out the new style of case discussion supported by the Zavic DB. It was evaluated favorably by many attendees, who were psychologically still dependent on the glass slide observation. Diagn. Cytopathol. 2013. © 2011 Wiley Periodicals, Inc.     

Accuracy of and reasons for frozen sections: a correlative, retrospective study

Previous studies on the accuracy of frozen sections (FS) were not based on the reasons for which the FS was performed. By omitting this important information, those studies reported FS accuracy of 94% to 97.4%. The current study analyzes the accuracy of FS diagnoses based on the reasons for which they were conducted. Of 482 specimens examined by FS in 1986, 41 were performed for evaluation of section margins of tumors, 29 for identification of unknown tissue, and 43 for detection of lymph node metastases. All 113 of these examinations proved to be 100% accurate. The remaining 369 FS were performed for diagnosis of an unknown pathologic process. Of these, 83.47% were precisely diagnosed; in 10.30%, the pathologic process was correctly, but not precisely, diagnosed; in 3.79%, the diagnosis was deferred; and the remaining 2.44% were incorrectly diagnosed (with no harmful consequences to the patients). By eliminating the cases of deferred diagnoses, the accuracy rate increased to 86.76%. Therefore, we suggest that the pathologist and the surgeon should not draw any conclusion from an inconclusive interpretation of FS and should proceed as though an FS had not been performed. When combining results of "precise diagnoses" with those of "correct pathologic process," the overall accuracy rate increased to 97%. The investigators conclude that FS should be used for a general diagnosis of a pathologic process rather than for an exact or precise diagnosis. Finally, we suggest that a similar survey of FS be periodically performed in every pathology department as part of its quality assurance program.