Repeatability and reproducibility of microscopic examination of adhesive tape strip cytology slides for the quantification of Malassezia spp. in canine skin.

BACKGROUND: The optimal microscopic magnification and number of optical fields of adhesive tape strip cytological slides that should be examined when searching for Malassezia yeasts on canine skin are unknown. OBJECTIVES: To determine the optimal magnification and the minimum number of optical fields that should be examined to maximise intraobserver repeatability and interobserver reproducibility. MATERIALS AND METHODS: Seven experienced examiners counted, twice, the number of yeasts in 10, 20, 30, 40 and 50 optical fields of 40 slides at ×400 and ×1000 magnification. RESULTS: The number of yeasts per unit surface area was significantly higher at ×1000 compared to ×400 magnification. Repeatability and reproducibility for counting the yeasts was very poor. CONCLUSIONS AND CLINICAL RELEVANCE: Adhesive tape strip cytological slides should be examined microscopically for Malassezia spp. at ×1000 magnification. The repeatability of this examination for counting the yeasts is poor.

Contexte - Le grossissement microscopique optimal et le nombre de champs optiques des lames cytologiques de bandes adhésives à examiner lors de la recherche de levures Malassezia sur la peau de chien sont inconnus. Objectifs - Déterminer le grossissement optimal et le nombre minimal de champs à examiner pour maximiser la répétabilité intra-observateur et la reproductibilité inter-observateur. Matériels et méthodes - Sept examinateurs expérimentés ont compté, deux fois, le nombre de levures dans 10, 20, 30, 40 et 50 champs de 40 lames aux grossissements ×400 et ×1 000. Résultats - Le nombre de levures par unité de surface était significativement plus élevé au grossissement ×1 000 par rapport au grossissement ×400. La répétabilité et la reproductibilité du comptage des levures étaient très médiocres. Conclusions et pertinence clinique - Les lames cytologiques de bandes adhésives doivent être examinées au microscope pour Malassezia spp. à un grossissement ×1 000. La répétabilité de cet examen de comptage des levures est faible.

Introducción- se desconoce el aumento microscópico óptimo y el número de campos ópticos de los portaobjetos citológicos en tiras de cinta adhesiva que deben examinarse al buscar levaduras Malassezia en la piel canina. Objetivos- determinar el aumento óptimo y el número mínimo de campos ópticos que deben examinarse para maximizar la repetibilidad intraobservador y la reproducibilidad interobservador. Materiales y métodos- siete examinadores experimentados contaron dos veces el número de levaduras en campos ópticos de 10, 20, 30, 40 y 50 de 40 portaobjetos con aumentos de x ×400 y ×1000. Resultados- el número de levaduras por unidad de superficie fue significativamente mayor con un aumento de ×1000 en comparación con un aumento de ×400. La repetibilidad y reproducibilidad para contar las levaduras fue muy pobre. Conclusiones y relevancia clínica - Los portaobjetos citológicos en tiras de cinta adhesiva deben examinarse microscópicamente para detectar Malassezia spp. con un aumento de ×1.000. La repetibilidad de este examen para contar las levaduras es pobre.

Contexto - A ampliação microscópica ideal e o número de campos ópticos das lâminas citológicas de fita adesiva que devem ser examinados nas pesquisas de leveduras do gênero Malassezia em cães são desconhecidos. Objetivos - Determinar a magnificação ideal e o número mínimo de campos ópticos que devem ser examinados para maximizar a repetibilidade intraobservador e a reproducibilidade interobservador. Materiais e métodos - Sete examinadores experientes contaram duas vezes o número de leveduras em 10, 20, 30, 40 e 50 campos ópticos de 40 lâminas nas magnificações de x400 e x1000. Resultados - O número de leveduras por unidade de área de superfície foi significativamente maior em x1000 em comparação com a ampliação de x400. A repetibilidade e a reprodutibilidade para a contagem de leveduras foi muito pobre. Conclusões e relevância clínica - Lâminas de citologia por fica adesiva devem ser examinadas microscopicamente para Malassezia spp a uma magnificação de x1.000. A repetibilidade deste exame para contagem de leveduras foi pobre.

ACGME Milestones 2.0: why and what's new for cytopathology?

BACKGROUND: Primary stakeholders in the Accreditation Council for Graduate Medical Education (ACGME) Milestones Project are: ACGME, Residency Programs, Residents, Fellowship Programs, Fellows, and Certification Boards. The intent of the Milestones is to describe the educational and professional developmental trajectory of a trainee from the first stages of their postgraduate education through the completion of their clinical training. The Milestones 2.0 project includes changes made based on experience with Milestones 1.0. METHODS: The ACGME solicited volunteers to participate in the development of subspecialty Milestones 2.0. The workgroup was charged with reviewing/making any additions to the four "Harmonized Milestones", developing subspecialty specific milestones for the Patient Care and Medical Knowledge competencies, and creating a supplemental guide. The Milestones were finalized following review of input from an open comment period. RESULTS: The Cytopathology Milestones 2.0 will go into effect July 2021. They include additional subcompetencies in the 4 harmonized competency areas and cytopathology-specific edits to the patient care and medical knowledge subcompetencies. Although the number of subcompetencies has increased from 18 to 21, within each subcompetency, the number of milestone trajectories has decreased. Additionally, within each subcompetency, the wording has been streamlined. A supplemental guide was created and Milestones 1.0 were compared to 2.0; however, curriculum mapping has been left to programs to develop. CONCLUSIONS: The ultimate goal of the Cytopathology Milestones 2.0 is to provide better real-time documentation of the progress of cytopathology fellows. The expected outcome is to produce highly competent cytopathologists, improving the care they provide, regardless of the program at which they trained.

Strand Orientation Bias Detector to determine the probability of FFPE sequencing artifacts.

Formalin-fixed paraffin-embedded tissue, the most common tissue specimen stored in clinical practice, presents challenges in the analysis due to formalin-induced artifacts. Here, we present Strand Orientation Bias Detector (SOBDetector), a flexible computational platform compatible with all the common somatic SNV-calling pipelines, designed to assess the probability whether a given detected mutation is an artifact. The underlying predictor mechanism is based on the posterior distribution of a Bayesian logistic regression model trained on The Cancer Genome Atlas whole exomes. SOBDetector is a freely available cross-platform program, implemented in Java 1.8.

Recommendations of the European Advisory Committee of Cytotechnology and European Federation of Cytology Societies for Training and Education of Cytotechnologists in Europe.

BACKGROUND: Faced with changes in cytodiagnostics, cervical cancer screening programs, the introduction and application of new methods, the cytotechnological educational program requires the necessary changes and additions. Insufficient, uneven as well as inaccessible education of cytotechnologists in European countries was the basis for making these recommendations. SUMMARY: The results of previous research and publications related to the currently available education of cytotechnologists in Europe, the needs and suggestions were given by the European Advisory Committee of Cytotechnology (EACC) and European Federation of Cytology Societies (EFCS) for optimal education of future generations of cytotechnologists were used in the preparation of these recommendations. The EACC and EFCS propose a 1-year education and training program divided into 3 modules: gynecological, nongynecological exfoliative, and fine-needle aspiration cytology. Training programs should be organized by an accredited university, preferably a combination of internal education in a cytology laboratory and theoretical education at the university. Cytopathologists and cytotechnologists with at least 5 years of work experience in cytodiagnostics should participate in education. Upon completion of the training program, the EACC and EFCS propose an official name: EFCS certified cytotechnologist. Key Messages: The EACC and EFCS believe that it is extremely important that these recommendations are recognized and implemented by institutions that provide education for cytotechnologists so that they can meet the growing requirements of the profession with their acquired knowledge and competencies.

Five-year retrospective review in gynecologic cytopathology: is it time to amend?

INTRODUCTION: According to the Clinical Laboratory Improvement Amendments 1988 regulations, 5-year retrospective review (5YRR) of normal Papanicolaou tests in patients with a newly diagnosed high grade squamous intraepithelial lesion or above (HSIL+) is mandatory. Since this mandate has been in place, a multitude of changes have taken place in the screening and management guidelines of cervical cancer. The aim of this study is to assess the role of this mandate in our laboratory and to investigate the lessons learned. MATERIAL AND METHODS: The cytopathology electronic database and institutional quality assurance records at Loyola University Medical Center were searched from January 2009 to December 2019 to identify all Papanicolaou tests diagnosed as new "HSIL and above" (HSIL+). Major discrepancy (2+) was defined as initial negative diagnosis changed to HSIL+. RESULTS: A total of 153,083 Papanicolaou tests were performed during this period; out of these, 1452 (0.94%) were diagnosed as HSIL+. A total of 695 HSIL+ Papanicolaou tests had a negative prior Papanicolaou and in 615 of 695 there was agreement with the initial negative diagnosis. In 61 Papanicolaou tests, the initial diagnosis was changed from negative and they were reclassified on review as 3 HSIL, 9 ASC-H, 7 AGC, and 42 ASCUS or LSIL. Major discrepancy rate was calculated as 3 of 695 (0.43%). None required an amended report. CONCLUSIONS: It is important to revisit the 5YRR as a method of implementing the quality indicators in gynecologic cytology so that the process retains its value without overburdening cytology laboratories and personnel.

The importance of cell culture parameter standardization: an assessment of the robustness of the 2102Ep reference cell line.

Work undertaken using the embryonic carcinoma 2102Ep line, highlighted the requirement for robust, well-characterized and standardized protocols. A systematic approach utilizing 'quick hit' experiments demonstrated variability introduced into culture systems resulting from slight changes to culture conditions (route A). This formed the basis for longitudinal experiments investigating long-term effects of culture parameters including seeding density and feeding regime (route B).Results demonstrated that specific growth rates (SGR) of passage 59 (P59) cells seeded at 20,000 cells/cm2 and subjected to medium exchange after 48h prior to reseeding at 72h (route B2) on average was marginally higher than, P55 cells cultured under equivalent conditions (route A1); whereby SGR values were (0.021±0.004) and (0.019±0.004). Viability was higher in route B2 over 10 passages with average viability reported as (86.3%±8.1) compared to route A1 (83.3±8.8). The metabolite data demonstrated both culture route B1 (P57 cells seeded at 66,667 cells/cm2) and B2 had consistent-specific metabolite rates (SMR) for glucose, but SMR values of route B1 was consistently lower than route B2 (0.00001 mmol, cell-1.d-1 and 0.000025).Results revealed interactions between phenotype, SMR and feeding regime that may not be accurately reflected by growth rate or observed morphology. This implies that current schemes of protocol control do not adequately account for variability, since key cell characteristics, including phenotype and SMR, change regardless of standardized seeding densities. This highlights the need to control culture parameters through defined protocols, for processes that involve culture for therapeutic use, biologics production, and reference lines.

Building a smart FNA cart: When Google meets cytology.

BACKGROUND: The appropriate management of a fine needle aspiration (FNA) supply cart and equipment set up is essential to ensure the smooth and optimal operation of a busy FNA clinic. We applied Lean strategies such as value stream mapping (VSM), the 5S method (Sort, Set in order, Shine, Standardize, Sustain), and Kanban to remove waste and improve patient flow in an FNA clinic. METHODS: The workflow analysis suggested that existent problems such as suboptimal inventory management and unavailability of standard operating procedures (SOPs) caused a 10% to 85% increase in total procedure time. To improve inventory management, we created a 2-bin Kanban system. We used the "Scan to Web" app and a Google Drive form to create a cost-effective electronic inventory management system. We distributed the essential SOPs in the format of video clips using our YouTube channel and leveraged barcode technology to access the links. RESULTS: Upon completion of our process improvement project, we succeeded to eliminate the stock-out events and maintain a process cycle efficiency of 87%. The 5S audit checklist result increased from 6% to 100% implementation, which is consistent with focused improvement. The developed inventory system enabled us to track the supply usage, forecast demands, and improve the accuracy of orders. CONCLUSIONS: Lean methods such as VSM, 5S, and Kanban combined with open source technologies can be implemented to ensure material availability, track inventory, and provide immediate access to SOPs on demand. The developed system also led to increased efficiency and improved flow, as well as responsiveness to changes in demand.

Bacterial mock communities as standards for reproducible cytometric microbiome analysis.

Flow cytometry has recently established itself as a tool to track short-term dynamics in microbial community assembly and link those dynamics with ecological parameters. However, instrumental configurations of commercial cytometers and variability introduced through differential handling of the cells and instruments frequently cause data set variability at the single-cell level. This is especially pronounced with microorganisms, which are in the lower range of optical resolution. Although alignment beads are valuable to generally minimize instrumental noise and align overall machine settings, an artificial microbial cytometric mock community (mCMC) is mandatory for validating lab workflows and enabling comparison of data between experiments, thus representing a necessary reference standard for the reproducible cytometric characterization of microbial communities, especially in long-term studies. In this study, the mock community consisted of two Gram-positive and two Gram-negative bacterial strains, which can be assembled with respective subsets of cells, including spores, in any selected ratio or concentration. The preparation of the four strains takes a maximum of 5 d, and the stains are storable with either PFA/ethanol fixation at -20 °C or drying at 4 °C for at least 6 months. Starting from this stock, an mCMC can be assembled within 1 h. Fluorescence staining methods are presented and representatively applied with two high-resolution cell sorters and three benchtop flow cytometers. Benchmarked data sets allow the use of bioinformatic evaluation procedures to decode community behavior or convey qualified cell sorting decisions for subsequent high-resolution sequencing or proteomic routines.

Immunocytochemistry practices in European cytopathology laboratories-Review of European Federation of Cytology Societies (EFCS) online survey results with best practice recommendations.

BACKGROUND: Variability in preanalytical and analytical steps for immunocytochemistry (ICC) on cytology samples is poorly defined. The objective of this study was to evaluate current practices for ICC on cytology samples in European laboratories. METHODS: A link to an online survey with 19 questions about ICC practices was distributed to cytology laboratories through national representatives in the European Federation of Cytology Societies. RESULTS: In total, 245 laboratories responded to the survey by January 30, 2019. Cell blocks, cytospins, liquid-based cytology (LBC) preparations, and smears alone or in combination with other preparations were used for ICC in 38%, 22%, 21%, and 19% of laboratories, respectively. In general, various combinations of preparations were used for ICC in greater than one-half of laboratories (147 of 245; 60%), whereas only 1 specific type of cytology preparation was used in the remaining 98 of 245 laboratories (40%) laboratories. The majority of laboratories (217 of 226; 96%) performed ICC on automated platforms using protocols that were the same as those used for formalin-fixed, paraffin-embedded samples (238 of 527 laboratories; 45%), either optimized (138 of 527 laboratories; 26%) or optimized and validated (151 of 527 laboratories; 29%) for cytology preparations. Positive control slides, negative control slides, and external quality control were used in 174 of 223 (78%), 112 of 223 (50%), and 111 of 120 (50%) laboratories, respectively. Greater than 1000 ICC tests were performed yearly in 34% of laboratories (65 of 191; average, 1477 tests; median, 500 tests). CONCLUSIONS: ICC is extensively performed in European laboratories using variously prepared cytology preparations on automated platforms, mostly without quality-assurance measures.

Cytologic and histologic samples from patients infected by the novel coronavirus 2019 SARS-CoV-2: An Italian institutional experience focusing on biosafety procedures.

The 2019 coronavirus pandemic, which started in Wuhan, China, spread around the globe with dramatic and lethal effects. From the initial Chinese epicenter, the European diaspora taxed the resources of several countries and especially those of Italy, which was forced into a complete social and economic shutdown. Infection by droplets contaminating hands and surfaces represents the main vehicle of diffusion of the virus. The common and strong efforts to contain the pandemic have relevant effects on the management of samples from histopathology laboratories. The current commentary reports and focuses on the protocols and guidelines in use at a large tertiary Italian hospital that accordingly are proposed for adoption in Italian laboratories as a potential model for national guidelines for the coronavirus emergency.

HDCytoData: Collection of high-dimensional cytometry benchmark datasets in Bioconductor object formats.

Benchmarking is a crucial step during computational analysis and method development. Recently, a number of new methods have been developed for analyzing high-dimensional cytometry data. However, it can be difficult for analysts and developers to find and access well-characterized benchmark datasets. Here, we present HDCytoData, a Bioconductor package providing streamlined access to several publicly available high-dimensional cytometry benchmark datasets. The package is designed to be extensible, allowing new datasets to be contributed by ourselves or other researchers in the future. Currently, the package includes a set of experimental and semi-simulated datasets, which have been used in our previous work to evaluate methods for clustering and differential analyses. Datasets are formatted into standard SummarizedExperiment and flowSet Bioconductor object formats, which include complete metadata within the objects. Access is provided through Bioconductor's ExperimentHub interface. The package is freely available from http://bioconductor.org/packages/HDCytoData.

Evaluating phase separation in live cells: diagnosis, caveats, and functional consequences.

The idea that liquid-liquid phase separation (LLPS) may be a general mechanism by which molecules in the complex cellular milieu may self-organize has generated much excitement and fervor in the cell biology community. While this concept is not new, its rise to preeminence has resulted in renewed interest in the mechanisms that shape and drive diverse cellular self-assembly processes from gene expression to cell division to stress responses. In vitro biochemical data have been instrumental in deriving some of the fundamental principles and molecular grammar by which biological molecules may phase separate, and the molecular basis of these interactions. Definitive evidence is lacking as to whether the same principles apply in the physiological environment inside living cells. In this Perspective, we analyze the evidence supporting phase separation in vivo across multiple cellular processes. We find that the evidence for in vivo LLPS is often phenomenological and inadequate to discriminate between phase separation and other possible mechanisms. Moreover, the causal relationship and functional consequences of LLPS in vivo are even more elusive. We underscore the importance of performing quantitative measurements on proteins in their endogenous state and physiological abundance, as well as make recommendations for experiments that may yield more conclusive results.

IAC standardized reporting of breast fine-needle aspiration cytology, Yokohama 2016: A critical appraisal over a 2 year period.

BACKGROUND: Breast cytology is a significant component of the "Triple approach" for pre-operative diagnosis of breast lumps, the other two being clinical assessment and radiological imaging. The role of Fine needle aspiration cytology (FNAC) as a first line investigation in diagnosing breast lesions is well documented, however histopathology is the gold standard. Cyto-histopathological correlation is of great relevance and also increases precision.AIMS \& OBJECTIVES:The present study was conducted with the aim to categorize breast lesions according to the latest standardized reporting system proposed by International academy of cytologists (IAC) in 2016. Evaluation of diagnostic accuracy, sensitivity and specificity of FNAC in diagnosing breast lesions and cyto-histopathological correlation was planned. MATERIALS AND METHODS: All FNAs of breast lesions over a period of 2 years were included in the study. The cases were grouped into five standardized categories proposed by the International academy of cytology: Category I (Insufficient material), Category II (Benign), Category III (Atypical, probably benign), Category IV (Suspicious, probably in situ or invasive) & Category V (Malignant) respectively. Specificity, sensitivity, diagnostic accuracy, negative and positive predictive value of FNAC were calculated and cyto-histopathological correlation assessed wherever possible. RESULTS: Out of 468 breast lesions reported on FNAC, the category wise distribution was - Category I, II, III, IV & V accounting for 23(4.9%), 342(73.07%), 7(1.5%), 11(2.35%) and 85(18.16%) respectively. Histopathology was performed in 331/468 cases with cyto histological concordance of 98.4% and a type agreement rate of 90.9%. The sensitivity, specificity, positive and negative predictive value and diagnostic accuracy was 98.90%, 99.16%, 97.82%, 99.58% and 99.09% respectively. CONCLUSION: FNAC is a simple, reliable, cost effective, first line diagnostic procedure for all breast lumps. In collaboration with physical examination and imaging studies (triple approach), FNAC is a highly sensitive diagnostic tool. Adopting a universally acceptable standardized reporting system for breast cytology can enhance the diagnostic accuracy of FNAC.

HPV testing for cervical cancer screening: technical improvement of laboratory logistics and good clinical performance of the cobas 6800 in comparison to the 4800 system.

BACKGROUND: European guidelines for cervical cancer screening now recommend the use of clinically validated assays for high-risk HPV-DNA sequences as primary test in women older than 30 years, performed in centralized laboratories, and run on systems providing automated solutions for all steps. METHODS: We conducted a comparison study, according to the international guidelines, nested within the organized population-based cervical screening program, between the cobas 4800 and 6800 systems (Roche Diagnostics), to evaluate accuracy and reproducibility of HPV test results and laboratory workflow. In Italy implementation of HPV cervical screening is under way on a regional basis; in Veneto it started in June 2015, following a piloting phase; the assay in use in the three centralized laboratories is the cobas 4800 HPV test, run on the cobas 4800 system. Comparison of HPV results with a new version of the assay (cobas 6800/8800 HPV) run on the cobas 6800 system, and intra- and inter-reproducibility analyses have been conducted in samples collected in PreservCyt medium (Hologic) from women without and with a subsequent diagnosis of high-grade lesion. RESULTS: Samples from women older than 30 years attending organized cervical cancer screening were used. Clinical sensitivity and specificity were evaluated on 60 cases and 925 controls, respectively; intra-laboratory reproducibility and inter-laboratory agreement by the 6800 system were evaluated on 593 and 460 specimens, respectively. Our results showed a very high agreement (> 98%) for overall qualitative results between the two systems; clinical sensitivity and specificity of the HPV assay run on 6800 were non-inferior to those of the HPV assay run on 4800 (p = 0,0157 and p = 0,0056, respectively, at the recommended thresholds of 90 and 98%); kappa values of 0.967 and 0.969 were obtained for intra- and inter-laboratory reproducibility analyses in the 6800 system. The 6800 platform displayed several technological improvements over the 4800 system, with higher throughput and laboratory productivity, and lower operator's hands-on time. CONCLUSIONS: The new cobas 6800/8800 HPV assay run on the 6800 instrument is suitable for use in large centralized laboratories included within population-based cervical cancer screening programs.

Importancia del sistema de Bethesda en el diagnóstico citológico de lesiones precancerosas del cérvix / The importance of Bethesda system in the cytological diagnosis of the cervix pre-cancerous lesions

RESUMEN El cáncer cérvico uterino, causa alrededor de 250 000 muertes anuales en el mundo y alrededor de 400 en Cuba, a pesar del esfuerzo que realiza el MINSAP, a través del Programa de Pesquisaje. Con el mismo se puede obtener el diagnóstico de lesiones precursoras del cáncer de cuello uterino, este diagnóstico citológico se realiza en Cuba a través del método de Richard y Barron que demuestra que existe un progreso citológico aparente hasta llegar al cáncer, que comienza con neoplasia intraepitelial (NICI a NICIII y carcinoma in situ), hasta finalmente el cáncer invasor. Por otro lado existe el método de Bethesda que responde casi todas las interrogantes que la citología plantea para su enfrentamiento, evidentemente los mayores aportes y revisiones se enfocan al manejo de las citologías atípicas de significado incierto, ya que no sólo presentan un mayor número de posibles evaluaciones, sino que representan el mayor porcentaje de citologías alteradas y la inclusión del VPH en las lesiones de bajo grado. En Cuba todavía se clasifica por el método de Richard y no se utiliza el Bethesda. Por la alta incidencia de esta entidad el propósito de este trabajo es emitir consideraciones sobre la implementación del sistema de Bethesda en el diagnóstico citológico de lesiones precancerosas del cérvix.

ABSTRACT The cervical-uterine cancer causes almost 250 000 death a year around the world and around 400 in Cuba in spite of the efforts made by the Public Health Ministry through the Screening Program. With it, the diagnosis of lesions that are predecessors of the cervical cancer could be reached. This cytological diagnosis is carried out through the Richard and Barron method, showing that there is an apparent cytological progress leading to the cancer that begins with intraepithelial neoplasia (NICI and NICIII and carcinoma in-situ) and ends in the invasive cancer. From the other hand there is the Bethesda methods answering to all the questions cytology ask for confronting it. Obviously the biggest contributions and reviews are focused in the management of the atypical cytologies with uncertain significance since they not only have a higher number of possible evaluations, but also represent the highest percent of the altered cytologies and the inclusion of the HPV in low grade lesions. The classification in Cuba is still made by the Richard method and the Bethesda one is not used. Due to the high incidence of this entity, the aim of this article is exposing considerations on the implementation of the Bethesda system in the cytological diagnosis of cervix pre-cancerous lesions.