Cell block practices in European cytopathology laboratories.

BACKGROUND: There are numerous methods and procedures described for the preparation of cell blocks (CBs) from cytological samples. The objective of this study was to determine current practices and issues with CBs in European laboratories. METHODS: A link to an online survey, with 11 questions about CB practices, was distributed to cytology laboratories via participants of United Kingdom National External Quality Assurance Service for Cellular Pathology Techniques and national representatives in the European Federation of Cytology Societies. RESULTS: A total of 402 laboratories responded completely (337/402, 84%) or partially (65/402, 16%) to the survey by February 4, 2022. The most common CB practice is embedding cell pellets using plasma and thrombin (23.3%), agar (17.1%), Shandon/Epredia Cytoblock (11.4%), HistoGel (7.9%), and Cellient (3.5%). Other methods such as CytoFoam, albumin, gelatin, Cytomatrix, and collodion bags are rarely used (1.0%, 0.7%, 0.7%, 0.3%, and 0.2%, respectively). CBs are also prepared from naturally occurring clots or tissue fragments (29.5%) and cells scraped from unstained or prestained smears (4.4%). The most frequent issues with the CBs in a daily cytology practice are low cellularity (248/402, 62%) and dispersed cells (89/402, 22%), regardless of the CBs preparation method or how the samples for embedding were selected. CONCLUSIONS: There is a great variability in CB practices in European laboratories with low cellular CBs as the main issue. Additional studies are mandatory to evaluate and improve performance and cellular yield of CBs.

Editorial: Optimizing Schools of Cytology: Discussions from the 2022 ASC/IAC Cytology Education Symposium, North American Strategies, and European Symbiosis.

This report highlights information and outcomes from the November 2022 ASC/IAC joint Cytology Education Symposium, an annual conference organized by the Cytology Programs Review Committee. The manuscript provides information on shared educational opportunities and practices for cytology students and other learners in anatomic pathology, discusses recruitment strategies for schools of cytology, conveys teaching resources, introduces perspectives on virtual microscopy and online learning, and transmits information about wellness of students in schools of cytology.

Optimizing schools of cytology: Discussions from the 2022 ASC/IAC Cytology Education Symposium, North American Strategies, and European Symbiosis.

This report highlights information and outcomes from the November 2022 ASC/IAC joint Cytology Education Symposium, an annual conference organized by the Cytology Programs Review Committee. The manuscript provides information on shared educational opportunities and practices for cytology students and other learners in anatomic pathology, discusses recruitment strategies for schools of cytology, conveys teaching resources, introduces perspectives on virtual microscopy and online learning, and transmits information about wellness of students in schools of cytology.

Optimization of pre-analytical and analytical steps for DNA and RNA analysis of fresh cytology samples.

BACKGROUND: Different cytology preparations can be used for molecular diagnostics, however the influence of pre-analytical and analytical steps on the results are not yet well defined. We aimed to determine optimal steps for efficient extraction of DNA and RNA from fresh cells for molecular diagnostics. METHODS: MCF7 and FaDu human cell lines, were used as a model to determine fresh cells storage conditions (temperature: 25°C, 4°C, -20°C, -80°C; duration: 0 h, 4 h, 12 h, 24 h, 48 h) and optimal nucleic acids extraction method. Besides, the minimal number of total cells and minimal percentage of mutated cells needed for successful extraction of nucleic acids and subsequent determination of present mutation were evaluated. RESULTS: Extraction of nucleic acids using spin columns yielded the highest quantity and quality of nucleic acids. Isolation of nucleic acids was feasible in all storage conditions, however higher temperature and longer duration of fresh cells storage were associated with lower quality of isolated nucleic acids and similar quantification cycle of housekeeping genes. Successful molecular testing was feasible with least 104 cells, while specific mutation was detected in as low as 5% of mutated cells. CONCLUSIONS: Our cell line model, mimicking fresh cytology samples, showed that quantity of extracted either DNA or RNA declined with higher temperatures and longer duration of storage but regardless of the storage conditions, we successfully detected both housekeeping genes and mutated gene using qPCR.

Podocyturia in Fabry disease: a 10-year follow-up.

BACKGROUND: Fabry disease (FD) is a rare X-linked disorder of sphingolipid metabolism that results in chronic proteinuric nephropathy. Podocytes are one of the most affected renal cells and play an important role in the development and progression of kidney disease. Detached podocytes found in urine (podocyturia) are considered as a non-invasive early marker of kidney injury; however, the dynamics of podocyte loss remains unknown. METHODS: In this 10-year follow-up study, podocyturia and other renal clinical data were evaluated in 39 patients with FD. From 2009 to 2019, podocyturia was assessed in 566 fresh urine samples from 13 male and 26 female FD patients using immunocytochemical detection of podocalyxin. RESULTS: Podocyturia (number of podocytes per 100 mL of urine) was found in 311/566 (54.9%) of the samples, more frequently (68.9 ± 21.9% versus 50.6 ± 25.9%; P = 0.035) and with higher values (364 ± 286 versus 182 ± 180 number of podocytes per gram of creatinine (Cr) in urine; P = 0.020) in males compared with females. The mean number of assessed samples for each patient was 14.5 (range 3-40) and the frequency of samples with podocyturia ranged from 0% to 100% (median 57%). Podocyturia was already present in 42.9% of patients <20 years of age and in 89.5% of normoalbuminuric patients. Podocyturia correlated with albuminuria (urine albumin:Cr ratio) (r = 0.20, P < 0.001) and a higher incidence and values of podocyturia were observed in patients with lower estimated glomerular filtration rate. CONCLUSIONS: Our data demonstrated that podocyturia is an early clinical event in the development of nephropathy. In addition, we found podocyturia to be a discontinuous event with wide variability.

Time-related changes in cell morphology and biomarker immunoreactivity for cells stored in a buffer-based cell medium.

OBJECTIVE: Buffer-based cell media (BBCM) are a valuable tool in the post-collection processing of cytology samples, though with poorly defined effects on cell properties. In this study, time-related changes in cell morphology and biomarker immunoreactivity were evaluated for cells stored at room temperature in a BBCM prepared with bovine serum albumin (BSA) and ethylene diamine tetraacetic acid (EDTA). METHODS: Cytospins were prepared at five consecutive 24-hour intervals (0, 24, 48, 72, 96) from three human cell lines (MCF7, SK-MEL-28, FaDu) suspended and stored in BBCM. Preservation of cell morphology was evaluated on Papanicolaou-stained cytospins from the percentages of apoptotic cells. Preservation of immunoreactivity was evaluated for cytokeratins, oestrogen receptors, Ki67, and melanoma markers from the percentages of cells positive for the corresponding immunocytochemical reactions. RESULTS: Cell morphology was well preserved for the majority of cells of the three lines stored for 24 and 48 hours (93%, 97%, 98% and 62%, 81%, 88%, respectively), while the majority of cells were apoptotic after 72 and 96 hours (70%, 47%, 39% and 77%, 70%, 59%, respectively). The immunoreactivity of cytokeratins remained unchanged during the entire 96 hours, while that of melanoma markers (S100, HMB45, Melan-A) decreased by 27%, 2%, and 3%, respectively. The immunoreactivity of oestrogen receptors and Ki67 decreased by 29% and 17% after the first 24 hours, and was completely lost after 96 hours. CONCLUSIONS: A BBCM with the addition of BSA and EDTA facilitates good preservation of cell morphology and immunoreactivity of biomarkers for up to 48 hours at room temperature.

Preservation of biomarkers immunoreactivity on cytospins protected with polyethylene glycol.

INTRODUCTION: The aim of this retrospective study was to evaluate the preservation of biomarkers immunoreactivity on cytospins protected with polyethylene glycol (PEG). METHODS: In two independent cytopathology laboratories, immunocytochemical reactions were retrospectively evaluated on methanol-fixed and PEG-protected cytospins stored at room temperature (RT) for different time periods and compared with immunocytochemical reactions on corresponding baseline methanol-fixed cytospins. Semi-quantitatively assessed immunoreactivity, using scores from 0 to 3, was considered reduced if two sequential scores were lowered by at least one point. RESULTS: Immunocytochemical reactions for 40 biomarkers with membrane (10), cytoplasmic (22) and nuclear (8) localisation were performed on 921 slides prepared from 183 cytological samples. For the majority of biomarkers (29/37, 78%), immunoreactivity on PEG-protected cytospins stored at RT remained unchanged in the first 12 months. Immunoreactivity for GFAP, p40 and hepatocyte antigen was monitored and remained unchanged for 1, 8 and 7 months, respectively. Partial or complete loss of immunoreactivity on PEG-protected cytospins stored for less than 12 months was found on a single sample out of the total evaluated for CD3 (1/7), CD30 (1/4), CD45 (1/10), CK5/6 (1/7), MelanA (1/7) and vimentin (1/7), while more frequent changes of immunoreactivity were found for Ki67 (4/7) and p63 (2/7). CONCLUSION: Immunoreactivity on cytospins protected with PEG and stored at RT is well-preserved for at least 12 months for the majority of biomarkers.

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.

Use of vimentin immunocytochemical staining for evaluation of atypical cells in voided urine samples.

BACKGROUND: Cytomorphology of exfoliated atypical reactive/repair renal tubular cells (RRTC) can resemble atypical urothelial cells thus suggesting a differential diagnostic question of urothelial neoplasia in urinary cytology. Vimentin expression has been shown in RRTC and used for differentiation from atypical urothelial cells. METHODS: The institutional computer database was searched for urinary cytology cases with vimentin immunocytochemical staining (2008-2012). Original cytopathological diagnoses based on cytomorphology and the results of vimentin immunostaining were compared to follow-up data, including histopathological diagnosis, subsequent urinary cytopathology reports, and clinical findings. RESULTS: Of the 42 cases with vimentin immunocytochemical staining, 33 were positive and 9 negative. Consequently, significant renal disease was found in 9/33 (27%) of vimentin positive cases and nehrolithiasis in 4/33 (12%) of vimentin positive and 1/9 (11%) of vimentin negative cases. Erythrocyturia of undetermined origin was diagnosed in nine cases (seven vimentin positive and two negative). Urinary cytology follow-up was negative in three vimentin positive cases. Urothelial carcinoma was found in 3/9 (30%) of vimentin negative cases. Thirteen patients were lost to follow-up. CONCLUSIONS: Vimentin immunocytochemical staining could be used as an ancillary method for evaluation of atypical cells in urinary specimens in selected cases with RRTC exhibiting cytological atypia. Unnecessary diagnostic procedures for evaluation of urothelial carcinoma could be avoided in vimentin positive cases and further diagnostic work-up for evaluation of a significant renal disease could be suggested in vimentin positive cases. Diagn. Cytopathol. 2017;45:85-90. © 2016 Wiley Periodicals, Inc.

Application of brush cytology for FISH-based detection of 1p/19q codeletion in oligodendroglial tumors.

Currently, oligodendroglial tumours (OT) are routinely tested for 1p/19q codeletion, a genetic abnormality of prognostic value. This analysis is most commonly performed by fluorescence in situ hybridization (FISH) on formalin-fixed, paraffin embedded (FFPE) tissue sections, which is time-consuming and often difficult to interpret, mainly due to the presence of truncated and overlapping nuclei. To overcome these methodological disadvantages, we investigated the validity of cytospins prepared from brushings of fresh tissue samples for assessing 1p/19q status by FISH. For this purpose, FISH analysis of 1p/19q codeletion was performed on FFPE tissue sections and cytospins prepared from brushing of corresponding fresh tissue in a series of 35 central nervous system tumours (16 OT and 19 non-OT). An aberrant 1p/19q status was found in 11/16 (69 %) OT samples and included codeletion of 1p/19q (7), 1p/19q imbalance (2) isolated 19q deletion (1) and 1p imbalance (1). None of the 19 non-OT samples showed 1p/19q codeletion. Results of FISH were concordant between FFPE sections and cytospins in all cases in which both types of slides gave interpretable results. Interpretation of FISH signals on cytospins was easier and quicker than on FFPE sections. Our study showed that cytospins prepared from brushing of fresh tissue samples allow quick and reliable FISH based analysis of 1p/19q status and can substitute traditional FFPE sections when fresh tissue is available.

Symptoms and signs of ocular surface disease related to topical medication in patients with glaucoma.

PURPOSE: The aim of this study was to assess signs and symptoms of ocular surface disease (OSD) and the cytomorphological changes of ocular surface in glaucoma patients using preserved antiglaucoma drops. METHODS: In this cross-sectional study, 109 participants (79 patients with topical medication and 30 untreated controls) completed the Ocular Surface Diseases Index (OSDI) questionnaire and underwent an ophthalmic examination, including Schirmer test, tear film breakup time (TBUT), and fluorescein staining. Conjunctival specimens were collected by impression cytology and analyzed by light microscopy using Nelson's grading scheme (grades 0-3). This classification is based on the nucleus-to-cytoplasm ratios of epithelial cells and the numbers of goblet cells, with grade 2 considered abnormal. RESULTS: The medication group had significantly shorter TBUT (median [interquartile range]: 6.0 seconds [5.0-8.0 seconds] vs 9.5 seconds [6.0-12.3 seconds]; P<0.03), greater fluorescein staining (1.0 [0.75-1.25] vs 0 [0-0.25]; P<0.001), and higher impression cytology grade than the control group (median [range]: 1.0 [1:2 to 1:6] vs 0.6 [1:2 to 1:4]; P<0.001). The increasing number of drops instilled per day was associated with an increase in fluorescein staining (Spearman's rho r=0.475; P<0.001) and shorter TBUT (r=-0.278; P=0.014). The OSDI did not discriminate between the two groups. CONCLUSION: Clinical tests and impression cytology showed ocular surface damage in patients using preserved antiglaucoma medications. However, there was no statistically and clinically significant difference in symptoms measured by OSDI score between the medication and control groups.