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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., (© 2022 ESVD and ACVD.)

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/cm 2  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/cm 2 ) 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.

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., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

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., (Copyright © 2021 American Society of Cytopathology. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2021 American Society of Cytopathology. Published by Elsevier Inc. All rights reserved.)

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., (© 2021 S. Karger AG, Basel.)

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., (© 2020 American Cancer Society.)

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., (© 2020 American Cancer Society.)

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.

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., (© 2020 American Cancer Society.)

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., (© 2019 McSwiggen et al.; Published by Cold Spring Harbor Laboratory Press.)

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., Competing Interests: No competing interests were disclosed., (Copyright: © 2019 Weber LM and Soneson C.)

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.

Introduction: MiRNAs are small endogenous non-coding RNAs implicated with gene expression regulation. Changes in miRNA levels have been reported in thyroid cancer. Fine-needle aspiration cytology (FNAC) is the most reliable tool for differential diagnosis of thyroid nodules., Methods: We have analyzed 174 FNAC from 168 patients with thyroid nodules for expression levels of 11 miRNAs (miRNA197; -187; -181b-3p; -181b-5p; -224; -181a; 146b; -221; -222; -155 and miRNA183) known to be up-regulated in cancer tissues compared to benign lesions. Expression of miRNAs was analyzed in FNA samples calculating the fold change of miRNA expression relative to normal thyroid tissue after normalization to an endogenous control., Results: In FNAC, miRNA expression was confirmed to be higher in malignant or suspicious for malignancy nodules compared to benign, only for miRNA146b, -222 and -221 (fold change expression ≥ 5)., Conclusion: In this study, we confirmed that a limited set of miRNAs can be used for the differential diagnosis of thyroid nodules.

Objective: Total quality management, which basically involves pre-analytical, analytical, and postanalytical phases, is relatively more difficult in cytopathology due to descriptive reports and subjective variability. The pre-analytical phase of total quality management constitutes a major burden of errors in the laboratory and it has not been widely studied in cytopathology except in cervical Pap smears. The present study was therefore conducted to study the pre-analytical phase over 5 years from April 2013 until May 2018 at an ISO 15189: 2012-certified cytopathology laboratory, in a resource-limited setting. It was also intended to study the level of satisfaction of the patients and the training of the professionals provided in the cytology lab so as to improve and maintain high quality standards., Methods: The study included all the documents relating to the quality program used in the last 5 years in a medical institute situated in the north sub-Himalayan region of India. All the data were recorded and analysed for pre-analytical analysis of the total quality management system., Results: In total, 20,130 samples were received for cytological investigation. The total number of errors which were detected in the cytopathology lab were 1,430, constituting 7.1% of the total investigations done. The pre-analytical phase errors comprised 57% of the total errors (815/1,430), analytical errors constituted 11% (157/1,430), while postanalytical errors represented 32% (458/1,430). Incompletely filled requisition forms constituted the most common pre-analytical error (38%). There was a gradual decrease in the errors with time, but with a sudden increase between January to June 2015 due to new technicians joining during that period., Conclusion: Pre-analytical quality management is an essential component for maintaining the quality and reducing the errors in the cytopathology lab. Documentation, continuous training, and maintenance of internal and external quality control with quality charts are the key for successful quality management. The identification of non-conformance with root-cause analysis and provision of scope for continuous improvement is vital for quality improvement in the lab. Patient satisfaction is an important aspect of quality in the lab, which should be combined with the satisfaction of the personnel working in the lab., (© 2018 S. Karger AG, Basel.)

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.

Clinical signs of prostatic diseases in dogs are often non-specific. Appropriate treatment should be based on a detailed investigation using reliable diagnostic tools. The aim of our study was to evaluate the diagnostic value of ultrasonography (US) and fine-needle aspiration (FNA) cytology in dogs' prostate diseases. The mean accuracy of FNA cytology and US were 0.72 and 0.88 (n = 13), respectively. US gland size measurements and actual gland dimensions were highly concordant. Obtained results confirm the high diagnostic value of US and FNA biopsy and in prostatic diseases. Diagnosis based on US is highly reliable; however, it should be combined with clinical signs. Therefore, cytological evaluation of prostate gland material may be performed to differentiate or confirm presumptive diagnosis., (© 2018 Blackwell Verlag GmbH.)

Background: Information on the performance of anal cytology in women who are high risk for human papillomavirus-related lesions and the factors that might influence cytology are largely lacking., Methods: Retrospective study including all new referrals of women with a previous history of anogenital neoplasia from January 2012 to July 2017, with concomitant anal cytology and high-resolution anoscopy with or without biopsies., Results: Six hundred and thirty six anal cytology samples and 323 biopsies obtained from 278 women were included. Overall sensitivity and specificity of "any abnormality" on anal cytology to predict any abnormality in histology was 47% (95% confidence interval [CI], 41%-54%) and 84% (95% CI, 73%-91%), respectively. For detecting high-grade squamous intraepithelial lesions (HSIL)/cancer, sensitivity was 71% (95% CI, 61%-79%) and specificity was 73% (95% CI, 66%-79%). There was a poor concordance between cytological and histological grades (κ = 0.147). Cytology had a higher sensitivity to predict HSIL/cancer in immunosuppressed vs nonimmunosuppressed patients (92% vs 60%, P = .002). The sensitivity for HSIL detection was higher when 2 or more quadrants were affected compared with 1 (86% vs 57%, P = .006). A previous history of vulvar HSIL/cancer (odds ratio [OR], 1.71, 1.08-2.73; P = .023), immunosuppression (OR, 1.88, 1.17-3.03; P = .009), and concomitant genital HSIL/cancer (OR, 2.51, 1.47-4.29; P = .001) were risk factors for abnormal cytology., Conclusions: Women characteristics can influence the performance of anal cytology. The sensitivity for detecting anal HSIL/cancer was higher in those immunosuppressed and with more extensive disease.

Context: - Pathology services are poorly developed in Sub-Saharan Africa. Komfo Anokye Teaching Hospital in Kumasi, Ghana, asked for help from the pathology department of the University Hospital of North Norway, Tromsø., Objective: - To reestablish surgical pathology and cytology in an African pathology department in which these functions had ceased completely, and to develop the department into a self-supporting unit of good international standard and with the capacity to train new pathologists., Design: - Medical technologists from Kumasi were trained in histotechnology in Norway, they were returned to Kumasi, and they produced histologic slides that were temporarily sent to Norway for diagnosis. Two Ghanaian doctors received pathology training for 4 years in Norway. Mutual visits by pathologists and technologists from the 2 hospitals were arranged for the introduction of immunohistochemistry and cytology. Pathologists from Norway visited Kumasi for 1 month each year during 2007-2010. Microscopes and immunohistochemistry equipment were provided from Norway. Other laboratory equipment and a new building were provided by the Ghanaian hospital., Results: - The Ghanaian hospital had a surgical pathology service from the first project year. At 11 years after the start of the project, the services included autopsy, surgical pathology, cytopathology, frozen sections, and limited use of immunohistochemistry, and the department had 10 residents at different levels of training., Conclusions: - A Ghanaian pathology department that performed autopsies only was developed into a self-supported department with surgical pathology, cytology, immunohistochemistry, and frozen section service, with an active residency program and the capacity for further development that is independent from assistance abroad.

Background: Although the correlations concerning cellular component analysis between the Sysmex XN-20 body fluid (BF) model and manual microscopy have been investigated by several studies, the extent of agreement between these two methods has not been investigated., Methods: A total of 90 BF samples were prospectively collected and analyzed using the Sysmex XN-20 BF model and microscopy. The extent of agreement between these two methods was evaluated using the Bland-Altman approach. Receiver operating characteristic (ROC) curve analysis was employed to evaluate the diagnostic accuracy of high-fluorescence (HF) BF cells for malignant diseases., Results: The agreements of white blood cell (WBC), red blood cell (RBC), and percentages of neutrophils, lymphocytes, and monocytes between the Sysmex XN-20 BF model and manual microscopy were imperfect. The areas under the ROC curves for absolute and relative HF cells were 0.67 (95% confidence interval [CI]: 0.56-0.78) and 0.60 (95% CI: 0.48-0.72), respectively., Conclusion: Due to the Sysmex XN-20 BF model's imperfect agreement with manual microscopy and its weak diagnostic accuracy for malignant diseases, the current evidence does not support replacing manual microscopy with this model in clinical practice., (© 2016 Wiley Periodicals, Inc.)

In this paper, we report a calibration of acoustic trapping force of single-beam acoustic tweezer (SBAT) at ultrahigh frequency using micropipette aspiration. The acoustic trapping forces ( F trapping ) and the trap stiffness on a 5- [Formula: see text] polystyrene microbead for a 110-MHz SBAT were measured against the known force generated from a micropipette. The trap stiffness ( k ), which represents F trapping corresponding to a displacement ( x ) of a microbead from the trap center, was measured and the results showed that a higher duty factor and excitation voltage lead to a stronger trapping force and trap stiffness for a given displacement. Since a precisely calibrated force generated from a micropipette is directly applied to the calculation of acoustic trapping force, the approach should be more flexible than those previously reported. In addition, with this method, precisely controlling the tip size of a micropipette within a few micrometers allows the possibility of calibrating the trapping force on an object of the size of a single cell. It not only helps better evaluate the trapping performance of SBAT as a tool of cell manipulation, but also helps develop SBAT as a useful tool for assessing cellular interactions.

A multi-technique approach was used to study the changes occurring in European eel Anguilla anguilla ovaries during hormonally-induced vitellogenesis. Aside from classic techniques used to monitor the vitellogenic process, such as ovary histology, fat content analysis, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and vitellogenin enzyme linked immunosorbent assay (ELISA), a new technique, Fourier-transform infrared (FT-IR) microspectroscopy, was used to analyse A. anguilla ovaries. The results from the different techniques provided different ways of approaching the same process. Although it is considered a time consuming approach, of all the employed techniques, histology provided the most direct evidences about vitellogenesis. SDS-PAGE and ELISA were also useful for studying vitellogenesis, whereas fat analysis cannot be used for this purpose. The FT-IR analysis provided a representative IR spectrum for each ovarian stage (previtellogenic stage, early vitellogenic stage, mid-vitellogenic stage and late vitellogenic stage), demonstrating that it is a valid method able to illustrate the distribution of the oocytes within the ovary slices. The chemical maps obtained confirmed changes in lipid concentrations and revealed their distribution within the oocytes at different maturational stages. When the results and the accuracy of the FT-IR analysis were compared with those of the traditional techniques commonly used to establish the vitellogenic stage, it became evident that FT-IR is a useful and reliable tool, with many advantages, including the fact that it requires little biological material, the costs involved are low, analysis times are short and last but not least, the fact that it offers the possibility of simultaneously analysing various biocomponents of the same oocyte., (© 2016 The Fisheries Society of the British Isles.)

Lineage tracing has become the method of choice to study the fate and dynamics of stem cells (SCs) during development, homeostasis, and regeneration. However, transgenic and knock-in Cre drivers used to perform lineage tracing experiments are often dynamically, temporally, and heterogeneously expressed, leading to the initial labeling of different cell types and thereby complicating their interpretation. Here, we developed two methods: the first one based on statistical analysis of multicolor lineage tracing, allowing the definition of multipotency potential to be achieved with high confidence, and the second one based on lineage tracing at saturation to assess the fate of all SCs within a given lineage and the "flux" of cells between different lineages. Our analysis clearly shows that, whereas the prostate develops from multipotent SCs, only unipotent SCs mediate mammary gland (MG) development and adult tissue remodeling. These methods offer a rigorous framework to assess the lineage relationship and SC fate in different organs and tissues., (© 2016 Wuidart et al.; Published by Cold Spring Harbor Laboratory Press.)

Objectives: Most non-screen-detected cervical cancers are advanced stage. We assess the potential for cytology to expedite diagnosis when used outside of routine call and recall screening for cervical cancer., Methods: Two cohorts of women with cytology that did not appear to have been taken as part of routine screening, nested within a census of cervical cytology, in England between April 2007 and March 2010 were studied: 93,322 women aged 40-69 at first cytology, and 14,668 women aged ≥70. The diagnostic performance of high grade cervical squamous intraepithelial lesion (HSIL) or worse cytology was estimated. We also estimated case-fatality from stage distribution in women aged ≥66 with and without cytology in the year prior to diagnosis., Results: There were 259 cancers diagnosed in women aged 40-69 at first cytology, and 78 in women aged ≥70. The sensitivity of cytology ≥ HSIL for cancer was 89% and 83% respectively, and the number of women needed to test to identify one cancer was 404 (95% confidence interval [CI]: 355-462) and 226 (95% CI: 177-292) respectively. Women aged ≥66 with cytology within a year of diagnosis had earlier stage cancers than those without, corresponding to a 17-22% reduction in case fatality., Conclusions: Cervical cytology is an excellent identifier of cancer among women tested outside routine screening call and recall. Its use as a triage tool, for instance in women with vague gynaecological symptoms, could facilitate earlier stage diagnosis and reduce cervical cancer mortality., (© The Author(s) 2015.)

Background: Brush cytology is the initial intervention when evaluating biliary strictures. Biliary brush cytology is known for its low sensitivity (but high specificity) and may be accompanied by biopsies and/or fluorescent in situ hybridization (FISH) to improve diagnostic yield. This study aimed to identify features to enhance cytological sensitivity, and assess which sampling method(s) improve identification of pancreatobiliary adenocarcinomas (PBCa)., Methods: Seventy-three biliary stricture cases were retrieved (38 PBCa and 35 control benign strictures). Biliary brushings, FISH, and biopsies were reviewed. Cytology specimens were evaluated for cellularity and presence of drunken honeycomb (DH), loosely cohesive clusters of round cells (LCCRC), large atypical cells with foamy cytoplasm (LACF), and single vacuolated malignant cells (SCs). Biopsies were examined for the presence of stromal invasion (SI)., Results: Biliary brushings were scantly cellular in 47.4% of PBCa and 51.4% of controls, resulting in 69.6% nondiagnostic/false-negative cytology diagnoses. DH, LACF, and SCs were significantly associated with adenocarcinoma (P < .00001, .0033, and .00002, respectively). By univariate analysis, SCs and LACF were predictors of malignancy in brushings (P = .0002 and .05). By multivariate analysis, only SCs were predictive of malignancy (P = .002). SI facilitated the diagnosis in 9 biopsies. Sensitivity/specificity of brush cytology, FISH, and biopsy were 39.5%/94.3%, 63.9%/94.3%, and 84.2%/100%, respectively., Conclusions: The low sensitivity of biliary brushings results from limited cellularity. Identification of LACF, DH, and SCs improves sensitivity. Sampling of stromal tissue may facilitate PBCa diagnosis. Concurrent biopsies and FISH are helpful in enhancing the diagnostic yield of PBCa., (© 2015 American Cancer Society.)

Background: Liquid-based cytology (LBC) for cervical screening would benefit from laboratory practice guidelines that define specimen adequacy for reporting of slides. The evidence base required to define cell adequacy should incorporate both ThinPrep™ (TP; Hologic, Inc., Bedford, MA, USA) and SurePath™ (SP; BD Diagnostics, Burlington, NC, USA), the two LBC systems used in the UK cervical screening programmes., Objectives: The objectives of this study were to determine (1) current practice for reporting LBC in England, Wales and Scotland, (2) a reproducible method for cell counting, (3) the cellularity of slides classified as inadequate, negative or abnormal and (4) the impact of varying cellularity on the likelihood of detecting cytological abnormalities., Design: The study involved four separate arms to pursue each of the four objectives. (1) A questionnaire survey of laboratories was conducted. (2) A standard counting protocol was developed and used by three experienced cytopathologists to determine a reliable and reproducible cell counting method. (3) Slide sets which included a range of cytological abnormalities were each sent to three laboratories for cell counting to study the correlation between cell counts and reported cytological outcomes. (4) Dilution of LBC samples by fluid only (unmixed) or by dilution with a sample containing normal cells (mixed) was performed to study the impact on reporting of reducing either the total cell count or the relative proportion of abnormal to normal cells., Setting: The study was conducted within the cervical screening programmes in England, Wales and Scotland, using routinely obtained cervical screening samples, and in 56 participating NHS cervical cytology laboratories., Participants: The study involved only routinely obtained cervical screening samples., Interventions: There was no clinical intervention., Main Outcome Measures: The main outcome measures were (1) reliability of counting method, (2) correlation of reported cytology grades with cellularity and (3) levels of detection of abnormal cells in progressively diluted cervical samples., Results: Laboratory practice varied in terms of threshold of cellular adequacy and of morphological markers of adequacy. While SP laboratories generally used a minimum acceptable cell count (MACC) of 15,000, the MACC employed by TP laboratories varied between 5000 and 15,000. The cell counting study showed that a standard protocol achieved moderate to strong inter-rater reproducibility. Analysis of slide reporting from laboratories revealed that a large proportion of the samples reported as inadequate had cell counts above a threshold of 15,000 for SP, and 5000 and 10,000 for TP. Inter-rater unanimity was greater among more cellular preparations. Dilution studies demonstrated greater detection of abnormalities in slides with counts above the MACC and among slides with more than 25 dyskaryotic cells., Conclusions: Variation in laboratory practice demonstrates a requirement for evidence-based standards for designating a MACC. This study has indicated that a MACC of 15,000 and 5000 for SP and TP, respectively, achieves a balance in terms of maintaining sensitivity and low inadequacy rates., Future Work: The findings of this study should inform the development of laboratory practice guidelines., Funding: The National Institute for Health Research Health Technology Assessment programme.

Context: Immunohistochemistry (IHC) is important for cytology but poses special challenges because preanalytic conditions may differ from the conditions of IHC-positive controls., Objective: To broadly survey cytology laboratories to quantify preanalytic platforms for cytology IHC and identify problems with particular platforms or antigens. To discover how validation guidelines for HER2 testing have affected cytology., Design: A voluntary survey of cytology IHC practices was sent to 1899 cytology laboratories participating in the College of American Pathologists Nongynecologic Cytopathology Education Program in the fall of 2009., Results: A total of 818 laboratories (43%) responded to the survey by April 2010. Three hundred fourty-five of 791 respondents (44%) performed IHC on cytology specimens. Seventeen different fixation and processing platforms prior to antibody reaction were reported. A total of 59.2% of laboratories reported differences between the platforms for cytology specimens and positive controls, but most (155 of 184; 84%) did not alter antibody dilutions or antigen retrieval for cytology IHC. When asked to name 2 antibodies for which staining conditions differed between cytology and surgical samples, there were 18 responses listing 14 antibodies. A total of 30.6% of laboratories performing IHC offered HER2 testing before publication of the 2007 College of American Pathologists/American Society of Clinical Oncologists guidelines, compared with 33.6% afterward, with increased performance of testing by reference laboratories. Three laboratories validated a nonformalin HER2 platform., Conclusions: The platforms for cytology IHC and positive controls differ for most laboratories, yet conditions are uncommonly adjusted for cytology specimens. Except for the unsuitability of air-dried smears for HER2 testing, the survey did not reveal evidence of systematic problems with any antibody or platform.

The terminology for reporting human papillomavirus-associated squamous lesions in the cervix, both in tissue samples and cytology specimens, has suffered from many changes throughout the last years creating confusion in interpreting cervical biopsy and cytology reports by clinicians. This review presents a summary and discussion of the current terminology for reporting results of cervical biopsies and cytology with emphasis in the lower anogenital squamous terminology consensus recommendations for tissue specimens and the 2001 Bethesda Workshop for reporting cytology results. Microscopic features of cervical lesions in tissue samples and cytology specimens are presented. Biomarkers, including p16 and Ki-67, are discussed and how they can help the pathologist when dealing with difficult cases.

Germ cells are the ultimate stem cells, and reports of their in vitro derivation generate excitement due to potential applications in reproductive medicine. To date, there is no firm evidence that meiosis, the hallmark of gametogenesis, can be faithfully replicated outside of the gonad. We propose benchmarks for evaluating in vitro derivation of germ cells, facilitating realization of their potential., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Insulin-like growth factor-I (IGF-I) is an important growth promoting protein that is involved in numerous cellular responses and multiple biological systems. Although the molecular structure, function and recombinant production of IGF-I in various hosts have been the subject of much researches over the recent past, methods to determine the bioactivity of this protein have not been fully explored. Several assays have traditionally been used to measure IGF-I bioactivity, but have not become a routine laboratory practice due to the high cost involved and technical problems. Thus, there is still a need for a rapid, technically simple and accurate assay to determine IGF-I bioactivity. This review highlights the various cell-based assays currently commercially available for measuring the bioactivity of IGF-I along with their limitations. This is aimed at presenting the modern-day IGF researcher with a holistic overview of the current trends and future prospects regarding IGF-I bioactivity determinations.

Background: Endometritis is a major cause of infertility in the mare. Therefore, the diagnosis of this disease is very important in veterinary practice. The objective of this study was to compare bacteriological and cytological results obtained from the mare uterus using biopsy (EB) and cytobrush (CB) techniques and relating these findings to the presence of polymorphonuclear cells (PMNs) in endometrial tissue as the gold standard for detection of endometritis. In particular, we tested the hypothesis that endometrial cytology and microbiology data obtained from material collected using the EB and CB techniques are similar, so that the CB technique could preferentially be used to detect subclinical endometritis in clinical practice., Methods: A total of 69 mares suspected of subclinical endometritis because of previous reproductive history and 15 maiden mares were enrolled in this study. Material collected from both EB and CB was smeared on sterile glass slides for cytological examinations and on culture media for microbiological examinations. Bacteriological cultures and cytological samples were classified as negative (no growth or mixed cultures of more than three microorganisms; <2% PMNs) or positive (pure growth of microorganisms; >2% PMNs) for endometritis., Results: Positive growth was observed in 43% of CB samples and in 54% of EB samples (difference not significant). The growth of β-hemolytic streptococci was always connected with positive cytology. This relationship was not observed for growth of E. coli or for non-pathogenic flora. The sensitivity of bacterial growth and cytology from EB was 0.63 and 0.73 respectively. The sensitivities of bacterial growth and cytology from CB were 0.50 and 0.71 respectively., Conclusion: Microbiological and cytological results obtained from CB are similar to those obtained from EB and based on these findings the CB technique may be recommended for collection of materials from the mare's uterus in clinical practice.

Background: Multi-cellular segmentation of bright field microscopy images is an essential computational step when quantifying collective migration of cells in vitro. Despite the availability of various tools and algorithms, no publicly available benchmark has been proposed for evaluation and comparison between the different alternatives., Description: A uniform framework is presented to benchmark algorithms for multi-cellular segmentation in bright field microscopy images. A freely available set of 171 manually segmented images from diverse origins was partitioned into 8 datasets and evaluated on three leading designated tools., Conclusions: The presented benchmark resource for evaluating segmentation algorithms of bright field images is the first public annotated dataset for this purpose. This annotated dataset of diverse examples allows fair evaluations and comparisons of future segmentation methods. Scientists are encouraged to assess new algorithms on this benchmark, and to contribute additional annotated datasets.

Diagnostic cytology is a useful, noninvasive test with practical foundations in high-quality medicine and applications to practice building. Cytology will generate practice revenue whether assessed in-house or sent to a clinical pathologist. Thorough in-house evaluation is adequate in some cases, but expert opinion is important in many cases. Specimen slides should at least be reviewed in-house for assessment of cellularity and potential artifacts before submission to a reference laboratory. Reference laboratories also provide special stains and advanced molecular diagnostics to help further characterize many neoplastic processes, search for organisms, identify pigments, and address other important aspects of the lesion., (Copyright © 2013 Elsevier Inc. All rights reserved.)

A careful calibration of the fluorescence setup is essential when using Fura-2 for quantitative fluorimetry. The calibration procedure described here is used for experiments in which the indicator dye is introduced into the cell through a patch-clamp pipette. The fluorescence ratio is determined in whole-cell recordings for three different calibration parameters that reflect different [Ca(2+)]: very high (R1), intermediate (Keff), and zero (R0).

Ca(2+) indicator dyes by necessity are Ca(2+) chelators, because it is the binding of Ca(2+) to dye molecules that induces the change in fluorescence on which the Ca(2+) signal is based. As chelators, once introduced into a cell, they contribute to cellular Ca(2+) buffering. It has been a question of much debate to what extent this added Ca(2+) buffer (exogenous Ca(2+) buffer) changes Ca(2+) homeostasis and the signals of interest. I discuss this problem here, emphasizing the distinction between the influence of the dyes on amplitudes (which may be not so severe) and on the dynamics of Ca(2+) signals (which may be drastic). Once the Ca(2+)-buffering action of dyes relative to intrinsic Ca(2+) buffers is understood for a given preparation, Ca(2+) dyes can be used as very versatile tools for studying both Ca(2+) concentrations and Ca(2+) fluxes. I describe in detail some of my own experiences in calibrating the indicator dye Fura-2. These refer exclusively to experiments in which the dye is loaded into the cell via a patch pipette because acetoxymethyl ester loading introduces problems that very often prohibit precise quantitative conclusions.

Gene therapy has proven its potential to cure diseases of the hematopoietic system, but potential adverse reactions related to insertional mutagenesis by integrating gene vectors and chromosomal instability in long-lived repopulating cells have emerged as a major limitation. Preclinical gene therapy in murine models is a powerful model for assessment of gene marking efficiency and adverse reactions. However, changes in the hematologic composition after transplantation with retrovirally modified hematopoietic stem cells have not been well investigated in large cohorts of animals by systematic cytological analyses. In the present study, cytological analyses of bone marrow and spleen were performed in a large cohort (n = 58) of C57BL/6J mice over an extended observation period after gene marking. Interestingly, we observed hematological malignancies in four out of 30 animals transplanted with dLNGFR (truncated form of the human p75 low-affinity nerve growth factor receptor) and tCD34 modified stem/progenitor cells. Our data demonstrate that cytological analysis provides important information for diagnosis of hematological disorders and thus should be included in preclinical studies and performed in each investigated animal. Together with histological analysis, flow cytometric analysis, and other analyses, the quality and predictive value of preclinical gene therapy studies will be improved.

The cytological analysis is an important diagnostic method in many clinical disciplines. In particular cases, the cytological and histologic analysis provides ultimate interpretation of observed form of human pathology, including the most intricate for clinical diagnostic cases of oncologic diseases. The verity of cytological and histologic conclusion about the results of analysis of tissue assay is based on the structural characteristics of tissues, cells and their components. All this, in its turn, significantly depends on stain applied In the laboratory medicine, the standardization provides settings of requirements to applied reagents with purpose to increase the validity of laboratory information. The project of national standard of the Russian Federation is presented founded on ISO 19001 regulating information provided by manufacturer with diagnostic reagents in vitro for staining in biology i.e. in cytological and histochemical analysis.

Mucous cell size and distribution were investigated in the skin of five salmon using a novel stereology-based methodology: one (48 cm) fish to test 15 tissue treatment combinations on measures of cell area and density on the dorsolateral region and, using the most suitable treatment, we mapped mucous cell differences between body regions on four (52 cm) salmon, comprising a male and a female on each of two diets. The section site, decalcification, embedding medium and plane of sectioning all impacted significantly on mucous cell size, whereas mucous cell density is more robust. There were highly significant differences in both mucosal density and mean mucous cell size depending on body site: the dorsolateral skin of the four salmon had significantly denser (about 8% of skin area) and larger (mean about 160 μm(2)) mucous cells, whereas the lowest mean density (about 4%) and smallest mean area (115 μm(2)) were found on the head. We found that 100 random measurements may be sufficient to distinguish differences >7 μm(2) in mean mucous cell areas. The results further suggest that salmon exhibit a dynamic repeatable pattern of mucous cell development influenced by sex, diet and possibly strain and season., (© 2012 Blackwell Publishing Ltd.)

The new Achievable Standards, Benchmarks for Reporting, and Criteria for Evaluating Cervical Cytopathology, 3rd edn (ABC3) includes radical changes in the criteria for evaluating cervical cytology. First, they include a new mission statement 'the objective of cervical screening is to reduce cervical cancer incidence and mortality by screening with a high sensitivity for the detection of CIN2 or worse, whilst maintaining a high specificity'. Second, the original four performance measurement criteria where laboratories were examined further if they were below the 10th or above the 90th percentile has been changed to three and laboratories are only mandatorily examined if they fall below the 5th or above the 95th percentile. The old criteria related to the percentage of samples that were inadequate, the percentage of all adequate samples reported as moderate dyskaryosis or worse (equivalent to high-grade squamous intraepithelial lesion or cancer), the percentage of adequate samples reported as mild dyskaryosis or borderline (equivalent to low-grade squamous intraepithelial lesion or atypical squamous/glandular cells) and the positive predictive value. The new criteria are percentage of inadequate samples, positive predictive value and a new measure termed referral value. These changes mean that far fewer laboratories will require mandatory examination. Third, a raft of optional performance measures have been introduced to help laboratories examine their annual statistical return to the Department of Health in comparison with other laboratories. These measures have been designed to produce a more uniform national programme, and to help laboratories decide whether they are maximizing the benefit of screening while minimizing the harm, which is the goal of all screening programmes. This review examines in detail the new criteria and explains in more detail some of the thinking behind them., (© 2012 Blackwell Publishing Ltd.)