BestCyte® primary screening of 500 ThinPrep Pap Test thin-layers: 3 Cytologists' Interobserver diagnostic concordance with predicate manual microscopy relative to Truth Reference diagnoses defining NILM, ASCUS+, LSIL+, and ASCH+ thresholds for specificity, sensitivity, and equivalency grading.

Background: The BestCyte® Cell Sorter Imaging System (BestCyte) facilitates algorithmic discrimination of clinically relevant cells in Pap test cytopathology by classifying and projecting images of cells in galleries based on cytomorphology. Warranted is awareness of potential BestCyte advantages as measured through 3 cytologists' interobserver diagnostic concordance, specificity and sensitivity differentials, and equivalency grading relative to manual microscopy (MM). Objectives: Using 500 MM-reported ThinPrep thin-layers, analyze: (1) cytologists' blinded BestCyte screening to raise Bethesda diagnoses; (2) correlate BestCyte and MM diagnoses (i.e., predicate) to establish Truth Reference Diagnoses (TRDx) from concordance between 4 possible diagnoses; (3) analyze cytologists' and MM predicate diagnoses through 4 diagnostic thresholds defined by TRDx: NILM (Negative) for specificity, and ASCUS+, LSIL+, and ASCH+ (Positive) for graded sensitivity (with abnormal cells decreasing in size with increasing dysplasia); and, (4) statistically determine cytologists' equivalency grading to MM using 95% Confidence Interval (CI) ranges. Results: 500 TRDx breakdown (n/%): NILM (241/48.2), ASCUS (79/15.8), ASCH (9/1.80), AGUS (2/0.40), LSIL (86/17.2), HSIL (68/13.6), CA (2/0.40), UNSAT (13/2.60). TRDx breakdown (n/%) per 4 of 4, 3 of 4, 2 of 4 diagnostic concordances: 264 (52.8%), 182 (36.4%), 54 (10.8%), respectively. No cases of discordant diagnoses were recorded. HSIL TRDx were established from 66.2% of 4 of 4 concordances, followed by NILM (59.3%), LSIL (46.5%), ASCUS (41.8%); antithetically, from 4.40% of 2 of 4 concordances. Specificity for MM predicate (NILM): 67.08%; for Cytologists 1, 2, and 3: 89.71%, 82.30%, 97.53%, respectively. For NILM threshold, cytologists revealed Significantly Superior equivalency to MM. Sensitivity for ASCUS+, LSIL+, and ASCH+ thresholds: MM (91.36%, 86.67%, 74.36%); Cytologist 1 (95.88%, 96.97%, 94.87%); Cytologist 2 (95.47%, 95.76%, 93.59%), Cytologist 3 (94.65%, 95.15%, 98.72%), respectively. Cytologists revealed Significantly Superior equivalency to MM for graded Positive thresholds; with Cytologist 3 for ASCUS+ being: Superior. Conclusions: BestCyte detects and efficiently displays abnormal cells in strategic galleries standardizing objectivity by systematizing mosaics of cell-types for cytologists' consideration. BestCyte fosters consistent, enhanced cytologists' sensitivity values for the ASCUS+, LSIL+, and ASCH+ Positive thresholds relative to MM. Also, BestCyte facilitates improved specificity and superior equivalency grading to MM reflecting efficient screening, and reduced labor. Confident interpretations of small dysplastic epithelial cells characteristic of HSIL led to exceptional interobserver diagnostic concordance inferring BestCyte is primed for effective cervical cancer screening practice.

BestCyte® Cell Sorter Imaging System: Primary and adjudicative whole slide image rescreening review times of 500 ThinPrep Pap test thin-layers - An intra-observer, time-surrogate analysis of diagnostic confidence potentialities.

Background: The novel Artificial Intelligence-driven BestCyte® Cell Sorter Imaging System (BestCyte) enables hybrid digital screening through classification and sorting of tiles depicting cells in 8 galleries or whole slide image (WSI) reviews. Objectives: (1) Analyze expenditures of time (minutes) for primary BestCyte cell sorter screening and adjudicative WSI rescreening of 500 blinded, randomized ThinPrep thin-layers to determine review times per Bethesda nomenclature; (2) Analyze review times for NILM qualifier diagnoses reflecting increasing interpretive complexity (i.e., Inflammation, Reactive/Repair, Bacterial cytolysis, Bacterial vaginosis, Atrophy, and Atrophic vaginitis); (3) Challenge accuracy of primary diagnoses (Downgraded, Upheld, and Upgraded) following adjudicative WSI rescreening to assess correlated review times as surrogate indicators of diagnostic confidence in BestCyte functionality (i.e., learning curve); and (4) Correlate primary and adjudicative diagnoses to calculate intra-observer reproducibility Kappa coefficients per Bethesda nomenclature. Results: Of 500 thin-layers, the mean [primary/adjudicative rescreening review times (minutes)] were: Overall study [1.38/3.94], NILM [1.23/3.02], ASCUS [1.18/2.53], ASC-H [1.73/4.86], AGUS [1.84/6.34], LSIL [1.49/4.16], HSIL [1.52/4.10], CA [0.65/2.57]. Of 500 primary Bethesda diagnoses: 2 (0.40%) downgraded; 483 (96.6%) upheld; 15 (3.00%) upgraded after adjudicative WSI rescreening. Of 354 NILM diagnoses: 0 downgraded; 344 (97.2%) upheld; 10 (2.82%) upgraded. Of 34 ASCUS diagnoses: 2 (5.88%) downgraded; 28 (82.4%) upheld; 4 (11.8%) upgraded. Of 17 ASC-H diagnoses: 0 downgraded; 16 (94.1%) upheld; 1 (5.88%) upgraded. Of AGUS (n=1), LSIL (n=24), HSIL (n=52), CA (n=1), UNSAT (n=17): 100% upheld. Kappa coefficients with 95% (Confidence Intervals): Overall study 0.9305 (0.8983-0.9627), NILM 0.9429 (0.9110-0.9748), ASCUS 0.8378 (0.7393-0.9363), ASC-H 0.9112 (0.8113-0.9999), AGUS 1.0 (1.0-1.0), LSIL 0.9189 (0.8400-0.9978), HSIL 0.9894 (0.9685-0.9999), CA 1.0 (1.0-1.0), UNSAT 1.0 (1.0-1.0). Primary BestCyte cell image review time trends for NILM, ASCUS, LSIL, and HSIL, revealed plateaus relative to decreasing respective adjudicative WSI rescreening times. Conclusions: Given innovative robustness, BestCyte accommodates interpretive fundamentals, enabling shorter ThinPrep thin-layer review times with optimal intra-observer concordance per Bethesda nomenclature through classifying, ranking, sorting, and displaying clinically relevant cells efficiently in galleries. BestCyte fosters continuously optimizing diagnostic confidence learning curves; may supplant manual microscopy for primary screening.

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.

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.

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.

Significance, cytomorphology of decoy cells in polyomavirus-associated nephropathy: Review of clinical, histopathological, and virological correlates with commentary.

Human polyomaviruses (PyV) are ubiquitous, remaining predominantly inactive hence asymptomatic in the healthy, immunocompetent population. BK and JC PyV potentially infect pan-urinary tract epithelial cells. With reactivation, PyV disrupt cell cycling mechanisms, facilitating viral replication leading to cell necrosis, exfoliation, and, infrequently, carcinogenesis. Exfoliated PyV-infected cells pose diagnostic pitfalls, hence they are termed "decoy cells" as they may mimic high-grade urothelial carcinoma cells. BK polyomavirus-associated-nephropathy (BKVAN) is an inflammatory disease causing interstitial fibrosis with tubular atrophy in renal transplant recipients, increasing risk of graft loss. BKVAN is confirmed by renal biopsy, and managed by immunosuppression modulation. As voided urine may provide pan-reno-urinary tract sampling, cytopathology may serve a critical diagnostic purpose coupled with decoy cell quantification and indirect BK PyV load gauging. Thus, identification of decoy cells and differentiation from high-grade urothelial carcinoma cells, and degenerated, benign urothelial cells, is clinically essential. PyV virology and pathobiology in the context of renal transplantation, immuno-suppression and BKVAN, and, decoy cell cytomorphology and cytopreparation with commentary are highlighted. Decoy cell overall characteristics: variable degeneration; cytomegaly; comet-like shapes; angular cytoplasmic extensions; eccentric, polar nuclear placements; moderate anisocytosis; typically single cells with high N:C ratios. Cytoplasmic features: moderate-abundance; granular, blue-gray monochromatism. Nuclear features: karyomegaly; haphazardly-scattered chromatin densities; smudged, homogeneous, basophilic ground glass masses displacing chromatin alongside inner periphery of regular, symmetrical nuclear envelopes. Background features: granular cellular debris; inflammatory cells; intact and lyzed erythrocytes. Decoy cells lack coarse chromatin as in high-grade urothelial carcinoma cells. Benign urothelial cells exhibit low N:C ratios with fine chromatin distribution and euchromasia.

Lady Andromache (Mary) Papanicolaou: The Soul of Gynecological Cytopathology.

This commentary serves to impress the astonishing and selfless contributions that Andromache (Mary) Papanicolaou made toward the scientific development of the Papanicolaou (Pap) test; for she alone was, and remains, the hidden soul of gynecological cytopathology. The odyssey assumes even greater significance as 2014 marks 100 years since the beginning of this saga at Cornell Medical College. An awareness of the background historical details and events is important and relevant to this commentary. Her kindness, thoughtfulness, and devotion to her husband, Dr. George N. Papanicolaou, and his work, shall survive her indefinitely through the Pap test and through the women surviving cervical cancer globally as a result of their combined revolutionary scientific work.