IL-22 regulates inflammatory responses to agricultural dust-induced airway inflammation.

IL-22 is a unique cytokine that is upregulated in many chronic inflammatory diseases, including asthma, and modulates tissue responses during inflammation. However, the role of IL-22 in the resolution of inflammation and how this contributes to lung repair processes are largely unknown. Here, we tested the hypothesis that IL-22 signaling is critical in inflammation resolution after repetitive exposure to agricultural dust. Using an established mouse model of organic dust extract-induced lung inflammation, we found that IL-22 knockout mice have an enhanced response to agricultural dust as evidenced by an exacerbated increase in infiltrating immune cells and lung pathology as compared to wild-type controls. We further identified that, in response to dust, IL-22 is expressed in airway epithelium and in Ym1+ macrophages found within the parenchyma in response to dust. The increase in IL-22 expression was accompanied by increases in IL-22 receptor IL-22R1 within the lung epithelium. In addition, we found that alveolar macrophages in vivo as well as THP-1 cells in vitro express IL-22, and this expression is modulated by dust exposure. Furthermore, subcellular localization of IL-22 appears to be in the Golgi of resting THP1 human monocytes, and treatment with dust extracts is associated with IL-22 release into the cytosolic compartment from the Golgi reservoirs during dust extract exposure. Taken together, we have identified a significant role for macrophage-mediated IL-22 signaling that is activated in dust-induced lung inflammation in mice.

Omega-6 and Omega-3 Fatty Acid-Derived Oxylipins from the Lipoxygenase Pathway in Maternal and Umbilical Cord Plasma at Delivery and Their Relationship with Infant Growth.

Omega-3 and omega-6 fatty acids are important for neonatal development and health. One mechanism by which omega-3 and omega-6 fatty acids exert their effects is through their metabolism into oxylipins and specialized pro-resolving mediators. However, the influence of oxylipins on fetal growth is not well understood. Therefore, the objective of this study was to identify oxylipins present in maternal and umbilical cord plasma and investigate their relationship with infant growth. Liquid chromatography-tandem mass spectrometry was used to quantify oxylipin levels in plasma collected at the time of delivery. Spearman's correlations highlighted significant correlations between metabolite levels and infant growth. They were then adjusted for maternal obesity (normal body mass index (BMI: ≤30 kg/m2) vs. obese BMI (>30 kg/m2) and smoking status (never vs. current/former smoker) using linear regression modeling. A p-value < 0.05 was considered statistically significant. Our study demonstrated a diverse panel of oxylipins from the lipoxygenase pathway present at the time of delivery. In addition, both omega-3 and omega-6 oxylipins demonstrated potential influences on the birth length and weight percentiles. The oxylipins present during pregnancy may influence fetal growth and development, suggesting potential metabolites to be used as biomarkers for infant outcomes.

Concentrations of fat-soluble nutrients and blood inflammatory compounds in mother-infant dyads at birth.

BACKGROUND: Perinatal inflammation adversely affects health. Therefore, aims of this IRB-approved study are: (1) compare inflammatory compounds within and between maternal and umbilical cord blood samples at the time of delivery, (2) assess relationships between inflammatory compounds in maternal and cord blood with birth characteristics/outcomes, and (3) assess relationships between blood and placental fat-soluble nutrients with blood levels of individual inflammatory compounds. METHODS: Mother-infant dyads were enrolled (n = 152) for collection of birth data and biological samples of maternal blood, umbilical cord blood, and placental tissue. Nutrient levels included: lutein + zeaxanthin; lycopene; α-, ß-carotene; ß-cryptoxanthin; retinol; α-, γ-, δ-tocopherol. Inflammatory compounds included: tumor necrosis factor-α, superoxide dismutase, interleukins (IL) 1ß, 2, 6, 8, 10. RESULTS: Median inflammatory compound levels were 1.2-2.3 times higher in cord vs. maternal blood, except IL2 (1.3 times lower). Multiple significant correlations existed between maternal vs. infant inflammatory compounds (range of r = 0.22-0.48). While relationships existed with blood nutrient levels, the most significant were identified in placenta where all nutrients (except δ-tocopherol) exhibited relationships with inflammatory compounds. Relationships between anti-inflammatory nutrients and proinflammatory compounds were primarily inverse. CONCLUSION: Inflammation is strongly correlated between mother-infant dyads. Fat-soluble nutrients have relationships with inflammatory compounds, suggesting nutrition is a modifiable factor. IMPACT: Mother and newborn inflammation status are strongly interrelated. Levels of fat-soluble nutrients in blood, but especially placenta, are associated with blood levels of proinflammatory and anti-inflammatory compounds in both mother and newborn infant. As fat-soluble nutrient levels are associated with blood inflammatory compounds, nutrition is a modifiable factor to modulate inflammation and improve perinatal outcomes.

Omega-3 Fatty Acid-Derived Resolvin D2 Regulates Human Placental Vascular Smooth Muscle and Extravillous Trophoblast Activities.

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.

Bioactive metabolites of OMEGA-6 and OMEGA-3 fatty acids are associated with inflammatory cytokine concentrations in maternal and infant plasma at the time of delivery.

BACKGROUND & AIMS: Inflammation is necessary for a healthy pregnancy. However, unregulated or excessive inflammation during pregnancy is associated with severe maternal and infant morbidities, such as pre-eclampsia, abnormal infant neurodevelopment, or preterm birth. Inflammation is regulated in part by the bioactive metabolites of omega-6 (n-6) and omega-3 (n-3) fatty acids (FAs). N-6 FAs have been shown to promote pro-inflammatory cytokine environments in adults, while n-3 FAs have been shown to contribute to the resolution of inflammation; however, how these metabolites affect maternal and infant inflammation is still uncertain. The objective of this study was to predict the influence of n-6 and n-3 FA metabolites on inflammatory biomarkers in maternal and umbilical cord plasma at the time of delivery. METHODS: Inflammatory biomarkers (IL-1ß, IL-2, IL-6, IL-8, IL-10, and TNFα) for maternal and umbilical cord plasma samples in 39 maternal-infant dyads were analyzed via multi-analyte bead array. Metabolites of n-6 FAs (arachidonic acid and linoleic acid) and n-3 FAs (eicosapentaenoic acid and docosahexaenoic acid) were assayed via liquid chromatography-mass spectrometry. Linear regression models assessed relationships between maternal and infant inflammatory markers and metabolite plasma concentrations. RESULTS: Increased plasma concentrations of maternal n-6 metabolites were predictive of elevated pro-inflammatory cytokine concentrations in mothers; similarly, higher plasma concentrations of umbilical cord n-6 FA metabolites were predictive of elevated pro-inflammatory cytokine concentrations in infants. Higher plasma concentrations of maternal n-6 FA metabolites were also predictive of elevated pro-inflammatory cytokines in infants, suggesting that maternal n-6 FA status has an intergenerational impact on the inflammatory status of the infant. In contrast, maternal and cord plasma concentrations of n-3 FA metabolites had a mixed effect on inflammatory status in mothers and infants, which may be due to the inadequate maternal dietary intake of n-3 FAs in our study population. CONCLUSIONS: Our results reveal that maternal FA status may have an intergenerational impact on the inflammatory status of the infant. Additional research is needed to identify how dietary interventions that modify maternal FA intake prior to or during pregnancy may impact maternal and infant inflammatory status and associated long-term health outcomes.

Something Smells Fishy: How Lipid Mediators Impact the Maternal-Fetal Interface and Neonatal Development.

Normal pregnancy relies on inflammation for implantation, placentation, and parturition, but uncontrolled inflammation can lead to poor maternal and infant outcomes. Maternal diet is one modifiable factor that can impact inflammation. Omega-3 and -6 fatty acids obtained through the diet are metabolized into bioactive compounds that effect inflammation. Recent evidence has shown that the downstream products of omega-3 and -6 fatty acids may influence physiology during pregnancy. In this review, the current knowledge relating to omega-3 and omega-6 metabolites during pregnancy will be summarized.

Virtual microscopy in cytotechnology education: Application of knowledge from virtual to glass.

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.

Successful integration of thyroid cytopathology and surgical pathology education in an E-module format.

Context: The shift to digital learning in medicine is well underway and in fact spurred by the COVID-19 pandemic. The didactic portion of our institution's cytotechnology (CT) education program is online and delivered to learners across the nation. With CT education elevating to the master's degree level, there is a need to expand cytologic correlation with surgical resection specimens. We also wanted to afford pathology residents the same. Methods: We developed an online cytologic-histologic correlation digital learning module (e-module) addressing thyroid fine needle aspirations (FNAs) and surgical thyroidectomy specimens which was administered as part of coursework in the CT education and pathology residency programs. The module was 35 min long and consisted of guided narration with both formative and summative interactive quizzes. After completion of the module, participants were invited to fill a brief survey comprised of multiple choice, Likert, and free response questions. This study was approved by the institutional review board. Results: The 29 respondents were comprised of 22 CT students and 7 residents. CT students had minimal experience thyroid pathology prior to the module; residents were mixed. Twenty-three (79.3%) ranked the highest tiers for learning cytopathology through this module, 24 (82.8%) for learning thyroid surgical pathology, and 25 (86.2%) for cytologic-histologic correlation. All respondents stated they would like similar activities in the future. Conclusions: Teaching cytology-histology correlation for thyroid in an electronic format was effective and well-received by participants. There is a demand for these activities among current learners, suggesting that expanding the available repertoire will be beneficial.

Intrauterine Transfer of Polyunsaturated Fatty Acids in Mother-Infant Dyads as Analyzed at Time of Delivery.

Polyunsaturated fatty acids (PUFAs) are essential for fetal development, and intrauterine transfer is the only supply of PUFAs to the fetus. The prevailing theory of gestational nutrient transfer is that certain nutrients (including PUFAs) may have prioritized transport across the placenta. Numerous studies have identified correlations between maternal and infant fatty acid concentrations; however, little is known about what role maternal PUFA status may play in differential intrauterine nutrient transfer. Twenty mother-infant dyads were enrolled at delivery for collection of maternal and umbilical cord blood, and placental tissue samples. Plasma concentrations of PUFAs were assessed using gas chromatography (GC-FID). Intrauterine transfer percentages for each fatty acid were calculated as follows: ((cord blood fatty acid level/maternal blood fatty acid level) × 100). Kruskal-Wallis tests were used to compare transfer percentages between maternal fatty acid tertile groups. A p-value < 0.05 was considered significant. There were statistically significant differences in intrauterine transfer percentages of arachidonic acid (AA) (64% vs. 65% vs. 45%, p = 0.02), eicosapentaenoic acid (EPA) (41% vs. 19% vs. 17%, p = 0.03), and total fatty acids (TFA) (27% vs. 26% vs. 20%, p = 0.05) between maternal plasma fatty acid tertiles. Intrauterine transfer percentages of AA, EPA, and TFA were highest in the lowest tertile of respective maternal fatty acid concentration. These findings may indicate that fatty acid transfer to the fetus is prioritized during gestation even during periods of maternal nutritional inadequacy.

QuPath Digital Immunohistochemical Analysis of Placental Tissue.

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.

Eye tracking in cytotechnology education: "visualizing" students becoming experts.

INTRODUCTION: This study reports the potential of eye-tracking technology in determining screening skills of cytotechnology (CT) students while examining digital images (DI). MATERIALS AND METHODS: Twenty-five static DI of gynecologic cytology specimens were serially displayed on a computer monitor for evaluation by 16 CT students and 3 cytotechnologists at 3 locations. During evaluation, participant's eye movements were monitored with a Mirametrix S2 eye tracker (iMotions, Boston, MA) and EyeWorks software (Eyetracking, Solana Beach, CA). Students completed the protocol at: Period1 (P1)-4 months, Period2 (P2)-7 months, Period3 (P3)-11 months during their 1-year training; and the cytotechnologists only once. A general linear mixed model was used to analyze the results. RESULTS: The proportion of agreement on interpretations for cytotechnologists, students during P1, and students during P3 were 0.83, 0.62, and 0.70 respectively. The mean task duration in seconds for cytotechnologists, students during P1, and students during P3 were 21.1, 34.6, and 24.9 respectively. The mean number of fixation points for cytotechnologists, students during P1, and students during P3 were 14.5, 52.2, and 35.3, respectively. The mean number of gaze observations of cytotechnologists, students during P1, and students during P3 on region of interest (ROI) 1 were 77.93, 181.12, and 123.83, respectively; and, ROI 2 were 38.90, 142.79, and 92.46, respectively. CONCLUSIONS: This study demonstrated that students had decreased time, number of fixation points, gaze observations on ROI, and increased agreement with the reference interpretations at the end of the training program, indicating that their screening skills were progressing towards the level of practicing cytotechnologists.

Fat-soluble nutrients and Omega-3 fatty acids as modifiable factors influencing preterm birth risk.

Preterm birth is a leading cause of child morbidity and mortality, so strategies to reduce early birth must remain a priority. One key approach to enhancing birth outcomes is improving maternal dietary intake. Therefore, the purpose of this review is to discuss mechanisms on perinatal status of fat-soluble nutrients (carotenoids, retinol, tocopherols) and omega-3 fatty acids and how they impact risk for preterm birth. Literature review demonstrates that maternal dietary intake and biological (blood and placental tissue) levels of fat-soluble nutrients during pregnancy may provide antioxidative, anti-inflammatory, and immunomodulatory health benefits. Omega-3 fatty acids also promote increased production of specialized pro-resolving mediators, subsequently mediating inflammation resolution. Combined effects of these nutrients support appropriate placental organogenesis and function. Consequently, fat-soluble nutrients and omega-3 fatty acids serve as strong influencers for preterm birth risk. As dietary intake remains a modifiable factor, future intervention would benefit from a focus on optimizing perinatal status of these specific nutrients.

Initial Assessments of E-Learning Modules in Cytotechnology Education.

BACKGROUND: Nine E-learning modules (ELMs) were developed in our program using Articulate software. This study assessed our cytotechnology (CT) students' perceptions on the content of the ELMs, and the perceived influence of the ELMs on students' performance during clinical rotations. SUBJECTS AND METHODS: All CT students watched nine ELMs before the related classroom lecture and group discussion. Following that, students completed nine preclinical rotation surveys. After their clinical rotations, students completed nine postclinical rotation surveys. RESULTS: Statements on the content of the ELMs regarding the quality of the video and audio, duration, navigation, and the materials presented, received positive responses from the majority of the students. While there were a few disagreements and neutral responses, most of the students responded positively saying that the ELMs better prepared them for their role, as well as helped them to better perform their roles during the clinical rotation. The majority of the students recommended developing more EMLs for cytology courses in the future. CONCLUSIONS: This study has given hope that the ELMs have potential to enhance our online curriculum and benefit students, within the United States and internationally, who have no easy access to cytology clinical laboratories for hands-on training.

Digital Applications in Cytopathology: Problems, Rationalizations, and Alternative Approaches.

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.

Interprofessional Education in Allied Health Using Virtual Technologies.

BACKGROUND: Interprofessional education (IPE) is becoming increasingly prevalent in health science education, with the goal of preparing students to work collaboratively in teams within the healthcare environment. Students in our cytotechnology and radiation therapy (RT) programs used virtual technologies to demonstrate their professions using case studies. The purpose of this activity was to see if our students' knowledge of each other's professions and educational technologies increased and if the students had a better understanding of how they would work together in a healthcare team. METHODS: Participants included four cytotechnology students and five RT students. All were given a presurvey to determine their level of knowledge about each other's profession. The cytotechnology students presented cases involving gynecologic and lung cancers using virtual microscopy and explained how they screen slides and interpret cellular changes. The RT students explained how they would treat these same patients using the Virtual Environment Radiotherapy Training system (VERT), showing the cytotechnology students how the beam is guided to the exact spot for treatment. After the IPE activity, all participants were given a post-survey to determine their levels of understanding. RESULTS: The results indicated that the IPE activity increased the level of understanding regarding each other's professions and how they each fit together in the role of patient care. CONCLUSIONS: IPE activities, even on a small scale with two professions in the same college, can improve knowledge and collaboration between professions. More of these activities should be conducted for effective healthcare teams and improved patient outcomes.

Inception and Development of the Papanicolaou Stain Method.

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.

Online education in cytotechnology programs: a pilot study.

INTRODUCTION: The University of Nebraska Medical Center's cytotechnology program has received requests for an on-line program. The purpose of this study is to demonstrate that on-line education with virtual microscopy (VM) achieves similar screening and interpretation skills as traditional teaching methods using light microscopy (LM). MATERIALS AND METHODS: The pilot phase was conducted using the first two courses in the program. The students were divided into two groups; traditional and virtual. The virtual group replaced LM with VM, while the traditional group utilized traditional teaching methods. At the end of the two courses, the virtual group was shown how to use LM and was given glass slide examinations. RESULTS: The mean of the female genital tract (FGT) screening quizzes and exams of the traditional group was 92.5; the mean for the virtual group was 86.8. For the respiratory tract (RT) course, the traditional group had a mean of 96 for their screening exams while the virtual group's was 85.3. The glass slide examinations (FGT Mean = 98, RT Mean = 95.3) given to the virtual group at the end of the pilot study demonstrated their ability to apply screening and interpretation skill learned from VM to LM. CONCLUSION: The study concludes that screening and interpretation skills of the traditional and virtual groups were similar. It appears possible to train students using VM as the sole method of teaching. The study will be extended to another cohort of students using the entire curriculum to further demonstrate the soundness of these results.

Utilization of virtual microscopy in cytotechnology educational programs in the United States.

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.

Investigation of scanning parameters for thyroid fine needle aspiration cytology specimens: A pilot study.

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.