Background:The use of virtual microscopy (VM) in clinical cytology has been limited due to the inability to focus through three dimensional (3D) cell clusters with a single focal plane (2D images). Limited information exists regarding the optimal scanning parameters for 3D scanning.Aims:The purpose of this study was to determine the optimal number of the focal plane levels and the optimal scanning interval to digitize gynecological (GYN) specimens prepared on SurePath™ glass slides while maintaining a manageable file size.Subjects and Methods:The iScanCoreo Au scanner (Ventana, AZ, USA) was used to digitize 192 SurePath™ glass slides at three focal plane levels at 1 μ interval. The digitized virtual images (VI) were annotated using BioImagene's Image Viewer. Five participants interpreted the VI and recorded the focal plane level at which they felt confident and later interpreted the corresponding glass slide specimens using light microscopy (LM). The participants completed a survey about their experiences. Inter-rater agreement and concordance between the VI and the glass slide specimens were evaluated.Results:This study determined an overall high intra-rater diagnostic concordance between glass and VI (89-97%), however, the inter-rater agreement for all cases was higher for LM (94%) compared with VM (82%). Survey results indicate participants found low grade dysplasia and koilocytes easy to diagnose using three focal plane levels, the image enhancement tool was useful and focusing through the cells helped with interpretation; however, the participants found VI with hyperchromatic crowded groups challenging to interpret. Participants reported they prefer using LM over VM. This study supports using three focal plane levels and 1 μ interval to expand the use of VM in GYN cytology.Conclusion:Future improvements in technology and appropriate training should make this format a more preferable and practical option in clinical cytology.

Objective: Cytodiagnoses of specific malignancies are enabled through analyses of abnormal nuclear chromatin and cytoplasmic features in stained cells. Aim: The objective of this work was to explore the inception, development, and chemistry of the Pap stain method introduced in 1942 by Dr. G.N. Papanicolaou. Study Design: To achieve this, we carried out a review of the English literature. Results: Between 1914 and 1933, Papanicolaou first analyzed vaginal squamous cells in guinea pigs and later in human vaginal fluid samples using hematoxylin and eosin with limited color reactions, correlating the cell-type morphology with endocrinology and histology. The 5-dye Pap stain method evolved through 2 salient phases. The first, between 1933 and 1942, saw the introduction of alcohol-ether fixation and aqueous waterblue staining to enhance cellular transparency, aiding the distinction of cervical cancer cells from benign cells, with quantitative and qualitative assessment of squamous cell maturity. The second phase, between 1942 and 1960, saw the introduction and refinement of various alcoholic cytoplasmic counterstaining schemes with orange G and EA (light green, Bismarck brown, eosin) and phosphotungstic acid, allowing wider ranges of polychromasia and further enhancing cellular visualization, facilitating the distinction of cell types and improving diagnostic confidence. Conclusions: Development of the Pap stain method followed specific historical and scientific events. The staining method evolved following incremental improvements in cellular transparency achieved through tailored cellular fixation and cytoplasmic staining using variable dye and pH combinations.

Objective: The aim of this work was to raise awareness of problems using digital applications for examining, teaching, and applying telecytology at King Abdulaziz Medical City (KAMC), Riyadh, Saudi Arabia; University of Nebraska Medical Center (UNMC), Omaha, NE, USA; and University of Pittsburgh Medical Center (UPMC), Pittsburgh, PA, USA. The objective was to rationalize problems and propose alternative digital approaches. Study Design: We sought to identify solutions to improve the following: (a) interpretive examination scores at KAMC for complex cytological templates (i.e., high-grade squamous intraepithelial lesions [HSIL]) when using static digital images (SDI) of cells in regions of interest (ROI); (b) visualization of cells in 3D clusters when teaching at UNMC using 2D and 3D whole-slide imaging (WSI); and (c) visualization of cells through streaming telecytology at UPMC. Results: Composite SDI (CSDI) improved test scores for complex interpretations (i.e., HSIL) by converging diagnostic criteria from multiple ROI. Multiplane focusing through z-stacked WSI facilitated the teaching of cytological entities characterized by 3D cell clusters and consultative telecytology through robotic cell analysis. Conclusions: Adequately visualized cytomorphology and multiplane focusing are essential for virtual cytopathology examinations, teaching, or consultative telecytology. Visualization of diagnostic criteria through 2D or 3D imaging is critical. Panoptiq panoramic WSI with integrated z-stacked video clips enables optimal applied telecytology.

Background:Virtual microscopy (VM) is a technology in which the glass slides are converted into digital images. The main objective of this study is to determine if cellular morphology, learned through virtual microscopy, can be applied to glass slide screening.Materials and Methods:A total of 142 glass slides (61 teaching and 81 practice) of breast, thyroid, and lymph node fine needle aspiration body sites were scanned with a single focal plane (at 40X) using iScanCoreo Au (Ventana, Tuscan, AZ, USA, formerly known as BioImagene, California, USA). Six students including one distant student used these digital images to learn cellular morphology and conduct daily screening. Subsequently, all the students were tested on 10 glass slides using light microscopy (LM). At the end of the study, the students were asked to respond to an online survey on their virtual microscopy experience. The glass slide screening test scores of the participating students who were taught through VM and tested on glass slides (VMLM group) were compared with the last three classes of students who were taught through LM and tested on glass slides (LMLM group).Results:A non-parametric statistical analysis indicated no difference (P= 0.20) in the glass screening test scores between VMLM (median = 93.5) and LMLM groups (median = 87). The survey indicated that the annotated teaching slides and access to the VM, off campus, were well appreciated by the students.Conclusions:Although the students preferred LM, they were able to apply the cytological criteria learned through VM to glass slide screening. Overall, VM was considered a great teaching tool.

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.

Background: QuPath is an open-source digital image analyzer notable for its user-friendly design, cross-platform compatibility, and customizable functionality. Since it was first released in 2016, at least 624 publications have reported its use, and it has been applied in a wide spectrum of settings. However, there are currently limited reports of its use in placental tissue. Here, we present the use of QuPath to quantify staining of G-protein coupled receptor 18 (GPR18), the receptor for the pro-resolving lipid mediator Resolvin D2, in placental tissue. Methods: Whole slide images of vascular smooth muscle (VSM) and extravillous trophoblast (EVT) cells stained for GPR18 were annotated for areas of interest. Visual scoring was performed on these images by trained and in-training pathologists, while QuPath scoring was performed with the methodology described herein. Results: Bland–Altman analyses showed that, for the VSM category, the two methods were comparable across all staining levels. For EVT cells, the high-intensity staining level was comparable across methods, but the medium and low staining levels were not comparable. Conclusions: Digital image analysis programs offer great potential to revolutionize pathology practice and research by increasing accuracy and decreasing the time and cost of analysis. Careful study is needed to optimize this methodology further.

Omega-3 fatty acids are important to pregnancy and neonatal development and health. One mechanism by which omega-3 fatty acids exert their protective effects is through serving as substrates for the generation of specialized pro-resolving lipid mediators (SPM) that potently limit and resolve inflammatory processes. We recently identified that SPM levels are increased in maternal blood at delivery as compared to umbilical cord blood, suggesting the placenta as a potential site of action for maternal SPM. To explore this hypothesis, we obtained human placental samples and stained for the SPM resolvin D2 (RvD2) receptor GPR18 via immunohistochemistry. In so doing, we identified GPR18 expression in placental vascular smooth muscle and extravillous trophoblasts of the placental tissues. Using in vitro culturing, we confirmed expression of GPR18 in these cell types and further identified that stimulation with RvD2 led to significantly altered responsiveness (cytoskeletal changes and pro-inflammatory cytokine production) to lipopolysaccharide inflammatory stimulation in human umbilical artery smooth muscle cells and placental trophoblasts. Taken together, these findings establish a role for SPM actions in human placental tissue.

Background:Our cytotechnology (CT) program has been utilizing virtual microscopy (VM) as an adjunct educational resource since 2011.Aims:The aim of this study was to identify the utilization of VM in other CT programs across the United States (US).Subjects and Methods:A cover letter was sent to the program directors of all accredited CT programs in the US (excluding our program), requesting their participation in an online survey. After 2 days, the participants were sent an online link to the survey. The survey results were analyzed using descriptive statistics.Results:There were a total of 25 respondents to the survey. Among the 25, three CT programs use VM. Two of the three programs have been using VM for <2 years while another program for “2–4” years. The respondents found that VM's side-by-side comparison feature helped to demonstrate differences between diagnoses and preparation methods, and VM helped to preserve the important slides by digitizing them. Respondents believed that teaching with glass slides was very important. The reasons for not using VM were that VM is expensive and time-consuming to incorporate into the program, and lack of manpower resources to create digitized teaching files.Conclusions:The CT programs that use VM found it to be a valuable educational tool. Even though many were not using VM, responses from the survey indicated they will likely use it in the future.