Development and characterization of primary cell culture from the spinal cord of Asian seabass, Lates calcarifer.

Asian seabass, Lates calcarifer, is one of the most important fish species in aquaculture. An attempt was made to develop a primary cell culture from the spinal cord of Lates calcarifer by the enzymatic and mechanical dissociation method. The primary cell culture was sub-cultured for 20 times in Leibovitz's L-15 medium with 20% fetal bovine serum (FBS) and 0.5 nM of human neurotrophin-3 at 28°C. The primary cell culture was cryopreserved at different passage levels and recovery of cells after long-term storage was estimated about 75-85%. The authenticity of origin of primary cell culture from L. calcarifer was confirmed by polymerase chain reaction assay using species-specific mitochondrial 12S rRNA primer. The primary cell culture was designated as seabass spinal cord cells (SBSC). The cells morphologically resembled the neurons due to their neural-like prolongations and star-like structure. Immunophenotypic analysis of the SBSC revealed that they are of neuronal origin. The SBSC were found to be highly susceptible to striped jack nervous necrosis virus (SJNNV) and infection in the cells was confirmed by RT-PCR. In conclusion, this is the first innovative euryhaline fish neuronal primary cell culture of L. calcarifer now available for neurophysiological and neurotoxicological studies.

Morphometric analysis and functional insights into the serotonergic system of Girardia tigrina (Tricladida, Platyhelminthes).

Using immunocytochemistry, serotonergic nerve elements were documented in the nervous system of the planarian Girardia tigrina. Serotonin-immunopositive components were observed in the brain, ventral, dorsal and longitudinal nerve cords, transverse nerve commissures connecting the nerve cords, and in the nerve plexus. Whole-mount preparations of G. tigrina were analyzed by fluorescent and confocal laser scanning microscopy. An essential quantitative morphometric measurement of serotonin-immunopositive structures was conducted in three body regions (anterior, middle, and posterior) of the planarian. The number of serotonin neurons was maximal in the head region. The ventral nerve cords gradually decreased in thickness from anterior to posterior body ends. Physiological action of exogenously applied serotonin was studied in G. tigrina for the first time. It was found that serotonin (0.1 and 1 µmol L-1) accelerated eye regeneration. The transcriptome sequencing performed for the first time for the planarian G. tigrina revealed the transcripts of the tryptophan hydroxylase (trph), amino acid decarboxylase (aadc) and serotonin transporter (sert) genes. The data obtained indicate the presence of the components of serotonin pathway in G. tigrina. The identified transcripts can take part in serotonin turnover and participate in the realization of biological effects of serotonin in planarians, associated with eyes regeneration and differentiation.

Comparison of Claudin-4, BerEP4, Carcinoembryonic Antigen and MOC31 in Serous Fluids Metastases Demonstrate High Sensitivity of Claudin-4 at Low Cellularity.

INTRODUCTION: Claudin-4 has been described as a highly sensitive immunocytochemical marker for detection of metastatic carcinoma cells in effusion cytology specimens. This study aims to challenge the performance of claudin-4 in different types of malignancies and low cellularity specimens, by comparison with other markers in a large cohort of carcinomatous effusion specimens. METHODOLOGY: Cell block preparations from peritoneal and pleural fluid specimens were retrieved, with malignant (carcinoma) diagnoses confirmed by review of hospital diagnosis code and pathology reports. Claudin-4, BerEP4, CEA, and MOC31 immunocytochemistry were performed and scored by expression proportion and intensity. Tumor cellularity was assessed for subgroup analysis of low cellularity specimens. RESULTS: Totally 147 specimens (70 pleural, 77 peritoneal) of 68 lung, 62 breast, 9 gynecological, and 7 gastrointestinal carcinomas were retrieved. The average proportion expression of claudin-4 was highest (89.6%, vs. CEA 40.5%, BerEp4 18.6%, MOC31 16.8%) and the percentage of strong expression was highest for claudin-4 (72.1%). Expression levels of claudin-4 were consistently higher than other markers in subgroups of all primary sites. The difference was more significant for low cellularity specimens. High (≥50%) proportion expression was seen for 96.61% of cases for claudin-4 (vs. BerEp4 8.77%, CEA 46.55%, MOC31 8.77%, p < 0.001). These factors contributed to a low concordance between claudin-4 and BerEp4, CEA and MOC31 (K = 0.010-0.043). CONCLUSION: Claudin-4 is more sensitive than CEA, BerEp4 and MOC31, suitable for low cellularity specimens of most types of metastatic carcinoma and is a robust immunocytochemical marker for carcinoma that can be used solitarily.

GABAA receptors and neuroligin 2 synergize to promote synaptic adhesion and inhibitory synaptogenesis.

GABAA receptors (γ-aminobutyric acid-gated receptors type A; GABAARs), the major structural and functional postsynaptic components of inhibitory synapses in the mammalian brain, belong to a family of GABA-gated Cl-/HCO3 - ion channels. They are assembled as heteropentamers from a family of subunits including: α (1-6), ß(1-3), γ(1-3), δ, ε, π, θ and ρ(1-3). GABAARs together with the postsynaptic adhesion protein Neuroligin 2 (NL2) and many other pre- and post-synaptic proteins guide the initiation and functional maturation of inhibitory GABAergic synapses. This study examined how GABAARs and NL2 interact with each other to initiate the formation of synapses. Two functionally distinct GABAAR subtypes, the synaptic type α2ß2γ2-GABAARs versus extrasynaptic type α4ß3δ-GABAARs were expressed in HEK293 cells alone or together with NL2 and co-cultured with striatal GABAergic medium spiny neurons to enable innervation of HEK293 cells by GABAergic axons. When expressed alone, only the synaptic α2ß2γ2-GABAARs induced innervation of HEK293 cells. However, when GABAARs were co-expressed with NL2, the effect on synapse formation exceeded the individual effects of these proteins indicating a synergistic interaction, with α2ß2γ2-GABAAR/NL2 showing a significantly greater synaptogenic activity than α4ß3δ-GABAAR/NL2 or NL2 alone. To investigate the molecular basis of this interaction, different combinations of GABAAR subunits and NL2 were co-expressed, and the degree of innervation and synaptic activity assessed, revealing a key role of the γ2 subunit. In biochemical assays, the interaction between NL2 and α2ß2γ2-GABAAR was established and mapped to the large intracellular domain of the γ2 subunit.

Assessment of Ki-67 Proliferative Index in Cytological Samples of Nodal B-Cell Lymphomas.

BACKGROUND: The Ki-67 proliferative index (PI) is part of the diagnosis of nodal B-cell lymphoma (nBCL), but its determination in cytological samples is not standardized. We aimed to establish an approach for the accurate determination of the Ki-67 PI in cytological slides to differentiate between indolent and aggressive nBCLs. METHODS: Patients diagnosed with nBCL by fine-needle aspiration biopsy and subsequent excision biopsy were included. Cell suspensions were prepared from biopsy samples for CD3/Ki-67 double immunocytochemical staining and flow-cytometric verification of lymphoma B-cell counts. The Ki-67 PI was assessed by manual counting and eyeballing in cytology and eyeballing in histology. The cut-off values for the differentiation between aggressive and indolent lymphomas were determined for each method. RESULTS: A strong correlation between manual and flow-cytometric counting of lymphoma B cells was confirmed (interclass correlation coefficient (IC coef.) = 0.78). The correlation of the Ki-67 PI determined in cytological and histological slides was also strong (IC coef. > 0.80). Histologically, 55 cases were classified as indolent and 31 as aggressive nBCLs. KI-67 PI cut-off values of 28.5%, 27.5%, and 35.5% were established for manual counting and eyeballing in cytology and eyeballing in histology, respectively, with high sensitivity and specificity. CONCLUSIONS: The Ki-67 PI, assessed by manual counting and eyeballing in cytological samples, accurately differentiates between indolent and aggressive nBCLs.

Salivary Gland Fine-Needle Aspiration: The Current and Future Landscape.

Fine-needle aspiration represents a valid tool for the diagnosis/management of salivary gland lesions. The past years assessed the lack of uniform diagnostic reports for salivary cytopathology leading to interpretative issues. In 2015, an international group of cytopathologists developed an evidence-based tiered classification system for reporting salivary gland fine-needle aspiration (FNA) specimens, the "Milan System for Reporting Salivary Gland Cytopathology" (MSRSGC). The present landscape of salivary cytology is represented by the growing adoption of the MSRSGC and the assessment of its diagnostic role. The future landscape is characterized by the increasing role of ancillary techniques for diagnostic and prognostic purposes.

In Vitro and In Vivo Analysis of Neuronal Polarity.

Neuronal development is characterized by the unidirectional flow of signal from the axon to the dendrites via synapses. Neuronal polarization is a critical step during development that allows the specification of the different neuronal processes as a single axon and multiple dendrites both structurally and functionally, allowing the unidirectional flow of information. Along with extrinsic and intrinsic signaling, a whole network of molecular complexes involved in positive and negative feedback loops play a major role in this critical distinction of neuronal processes. As a result, neuronal morphology is drastically altered during establishment of polarity. In this chapter, we discuss how we can analyze the morphological alterations of neurons in vitro in culture to assess the development and polarity status of the neuron. We also discuss how these studies can be conducted in vivo, where polarity studies pose a greater challenge with promising results for addressing multiple pathological conditions. Our experimental model is limited to rodent hippocampal/cortical neurons in culture and cortical neurons in brain tissues, which are well-characterized model systems for understanding neuronal polarization.

Utility of immunocytochemistry in diagnosing abdominopelvic washings from patients undergoing radical surgery for endometrial cancer.

Objective: This study aimed to explore the efficacy of immunocytochemistry in diagnosing abdominopelvic washings (APWs) and evaluate the superiority of cytology combined with immunocytochemistry over cytology alone. Material and Methods: Data on APW cytology and available cell blocks from patients who underwent radical surgery for endometrial cancer between January 2021 and December 2022 were reviewed. Cytology was re-evaluated according to a five-tier system. Immunocytochemistry analysis for targets such as Sry box transcription factor 1(SOX17), Paired box gene 2 (Pax-2) protein, Phosphatase and tensin (PTEN), and ß-catenin was performed on each case with non-negative cytology. Mismatch repair (MMR) protein and P53 immunocytochemistry analyses were performed using cell blocks from cases with abnormal MMR or P53 expression in their primary lesion. The accuracies of cytology combined with immunocytochemistry and cytology alone were calculated. Results: Overall, 126 patients were included in this study, 18 of whom demonstrated non-negative cytology of APW. Cell blocks were successfully prepared for 16 cases. SOX17 positivity was observed in 16 cases, including 1 of serous carcinoma, 1 of clear cell carcinoma, and 14 of endometrioid carcinoma (EC). Loss of Pax-2 and PTEN expression was observed in the APWs of the 14 patients with EC. MMR deficiency was noted in two patients with EC, and P53 mutation was noted in another two patients with EC. Compared with 10 metastatic carcinomas (10/18, 55.56%) diagnosed by cytology alone, 15 malignant APWs (15/18, 83.33%) were confirmed through combination cytology and immunocytochemistry. APWs were more likely to be observed in cases with more than half myometrial invasion than those with no or less than half myometrial invasion (P = 0.0067). The probability of malignant APW occurrence was slightly elevated in cases of EC exhibiting microcystic, elongated, and fragmented(MELF) infiltrative growth (P = 0.039). Conclusion: SOX17 is a useful Müllerian marker for distinguishing endometrial epithelium in APW. Loss of Pax-2 and PTEN expression offers evidence of metastatic endometrial carcinoma. Furthermore, positive APWs retained molecular features similar to primary lesions. The use of multiple immunocytochemical markers can effectively enhance the diagnostic efficiency of APWs.

Practical issues related to immunocytochemistry on cytological smears: Tips and recommendations.

OBJECTIVE: Immunocytochemistry (ICC) is essential for enhancing diagnostic accuracy and identifying markers for diagnosis, prognosis and targeted therapies. While cell blocks (CBs) are preferred for standardization and optimized staining, cytological smears are an alternative when CBs are unavailable. However, the literature on ICC protocols for smears is sparse. This review addresses preparation, fixation and protocols for nuclear and cytoplasmic antibodies on smears, drawing from our laboratory's experience. METHODS: We reviewed procedures for ICC on cytological smears using existing literature and practical insights from our laboratory. RESULTS: Commercially available antibodies were found to be reliable for ICC on smears if specimens are properly prepared and fixed. Protocols developed in our laboratory maintained antigenicity and provided clear staining results. CONCLUSIONS: Although ICC on CBs is the gold standard for standardization, cytological smears are a viable alternative when CBs are unavailable. Success in ICC on smears depends on proper preparation and fixation. This review offers practical protocols and insights to help laboratories optimize ICC on cytological smears. Further research and standardization are necessary to enhance reproducibility and reliability of ICC on smears. The practical information provided is based on personal experience in our laboratory.

Cytopathology and clinicopathological correlation of renal neuroendocrine neoplasms.

INTRODUCTION: There is a lack of documentation regarding cytopathology of renal neuroendocrine neoplasms (NENs) due to their rarity. MATERIALS AND METHODS: Five cytology cases were gathered from 3 institutes. RESULTS: Cohort consisted of 4 females and 1 male. Fine needle aspiration biopsy and touch preparation slides of core needle biopsy revealed cellular samples, composed of round, plasmacytoid, or columnar cells. Tumor cells were present in nested, acinar, 3D cluster, and individual cell patterns. Tumor cells in 3 cases exhibited uniformly round to oval small nuclei with inconspicuous nucleoli, finely granular chromatin, and smooth nuclear membranes, whereas 2 other cases showed pleomorphic nuclei with conspicuous nucleoli, nuclear molding, and irregular nuclear membranes. Tumor cells displayed pale or granular cytoplasm, with 1 case showing small vacuoles. Examination of cores and cell blocks demonstrated tumor cells in sheets, nests, or acini. All tumor cells were positive for neuroendocrine immunomarkers. Based on mitotic count, Ki-67 index and morphology, 3 tumors were graded as well-differentiated neuroendocrine tumor (WDNET) (1 grade [G] 3, 1 G2, 1 G1) and 2 as large cell neuroendocrine carcinoma. Deletion of 7q, 10q, and 19q was detected in WDNETs. Two patients with large cell neuroendocrine carcinoma and 1 with WDNET G3 underwent chemotherapy due to aggressiveness, whereas nephrectomy was performed for patients with WDNET G1 and 2 without metastasis. CONCLUSIONS: Cytopathological characteristics of renal NENs closely resemble those affecting other organs. Despite its rarity, renal NENs should be kept in mind when confronted with morphological resemblances to NENs, to prevent misdiagnosis and inappropriate therapeutic interventions.

Cytochemical and Histochemical Staining with Peptide Antibodies.

Peptide antibodies are particularly useful for immunocytochemistry (ICC) and immunohistochemistry (IHC), where antigens may denature due to fixation of tissues and cells. Peptide antibodies can be made to any defined sequence, including unknown putative proteins and posttranslationally modified sequences. Moreover, the availability of large amounts of the antigen (peptide) allows inhibition/absorption controls, which are important in ICC/IHC, due to the many possibilities for false-positive reactions caused by immunoglobulin Fc receptors, nonspecific reactions and cross-reactivity of primary and secondary antibodies with other antigens and endogenous immunoglobulins, respectively. Here, simple protocols for ICC and IHC are described together with recommendations for appropriate controls.

Expression of Tshb and Tshr in the ricefield eel Monopterus albus: Potential paracrine/autocrine roles in gonads.

Thyroid stimulating hormone (TSH), a glycoprotein synthesized and secreted from thyrotrophs of the pituitary gland, is composed of a glycoprotein hormone common alpha subunit (CGA) and a specific beta subunit (TSHB). The major biological function of TSH is to stimulate thyroidal follicles to synthesize and secrete thyroid hormones through activating its cognate receptor, the thyroid stimulating hormone receptor (TSHR). In the present study, polyclonal antisera against ricefield eel Tshb and Tshr were generated respectively, and the expression of Tshb and Tshr was examined at mRNA and protein levels. RT-PCR analysis showed that tshb mRNA was expressed mainly in the pituitary as well as in some extrapituitary tissues including the ovary and testis. Tshr mRNA was also expressed in a tissue-specific manner, with transcripts detected in tissues including the kidney, ovary, and testis. The immunoreactive Tshb signals in the pituitary were shown to be localized to the inner areas of adenohypophysis which are close to the neurohypophysis of adult ricefield eels. Tshb-immunoreatvie cells in the pituitary of ricefield eel larvae were firstly observed at hatching. The expression of immunoreactive Tshb and Cga was also detected in ricefield eel ovary and testis together with Tshr. In the ovary, immunoreactive Tshb, Cga, and Tshr were observed in oocytes and granulosa cells. In the testis, immunoreactive Tshb was mainly observed in Sertoli cells while immunoreactive Cga and Tshr were detected in germ cells as well as somatic cells. Results of the present study suggest that Tsh may be synthesized both in the ovary and testis locally, which may play paracrine and/or autocrine roles in gonadal development in ricefield eels.

Integrated On-Slide Positive Controls for Immunocytochemistry on Cytology Slides.

INTRODUCTION: Integrated on-slide positive controls are a standard quality assurance and quality control measure for immunohistochemistry on formalin-fixed paraffin-embedded tissue sections. They ensure identical analytical conditions for the control and patient samples. Our aim was to develop a procedure for preparing integrated on-slide positive controls for immunocytochemistry (ICC) on methanol-fixed cytospins. METHODS: Leftover diagnostic cytology samples with sufficient cells and confirmed expression of Calretinin, MOC31, TTF1, and hormone receptors were used as control samples. Cells from the control samples were deposited on the peripheral part of objective slides using standard cytocentrifuge equipment. Cytospins were immediately fixed in methanol for at least 30 min and then covered with polyethylene glycol (PEG). Completely dry and solid PEG was removed from the central part of the objective slides and stored at room temperature. Patient samples were subsequently added to the central part of a PEG-protected slide, with an appropriate positive control placed on the peripheral part, and then fixed in methanol. ICC was performed on the Ventana/Roche automated platform ULTRA, using optimized and validated protocols for TTF1, hormone receptors, and double immunostaining for Calretinin/MOC31. The quality of ICC reactions for both deposits on the same slide and potential cell carryover was evaluated retrospectively. RESULTS: In the period from October 2021 to December 2023, the majority of integrated positive controls (364/368, 99%) consistently exhibited unequivocally positive reactions for TTF-1 (n = 93), hormone receptors (n = 84), and double staining for Calretinin/MOC31 (n = 191), with easily interpretable ICC reactions on corresponding patient samples. No obvious carryover of cells from the control sample to the patient sample was observed during this period. CONCLUSION: A novel approach developed for preparing integrated on-slide positive controls for ICC on methanol-fixed cytospins using standard cytocentrifugation is low-cost and can be widely applied in diagnostic cytology laboratories. Simultaneous ICC procedures for the control and patient samples on the same slide ensure identical analytical conditions for both samples, providing the highest level of quality control while reducing costs. Interpreting both ICC reactions on the same slide is time-efficient and convenient.

"Immunocytochemistry in Cytology: Myth or Reality": Unraveling the Myth - Immunocytochemistry Applications in Thyroid Lesions.

BACKGROUND: Fine-needle aspiration cytology serves as an important preoperative diagnostic tool for thyroid nodules. Despite its excellent diagnostic accuracy, diagnoses based solely on morphological observation can be challenging. Therefore, various ancillary diagnostic techniques have been applied, including immunocytochemistry (ICC). This review discusses the application and evaluation of ICC in thyroid fine needle aspiration. SUMMARY: Currently, three immunostaining preparation methods are available for cytological materials: liquid-based cytology, cell block, and cell transfer. ICC proves valuable in scenarios such as tumour diagnosis, assessment of differentiation and grading of carcinomas, estimation of primary organs in metastatic carcinomas, and detection of gene abnormalities. However, ICC, while useful, is not as accurate as immunohistochemistry and is more difficult to evaluate. KEY MESSAGES: If the pitfalls and limitations are understood and effectively navigated, ICC could play a significant role in decreasing the non-diagnostic rate, thus leading to more accurate and valuable diagnoses and reductions in the re-aspiration rate.

Serotonergic elements in the nervous system of parasite of acipenserid fishes, Acrolichanus auriculatus (Digenea: Allocreadiidae).

The trematode Acrolichanus auriculatus is a widely distributed intestine parasite of acipenserid fishes. For the first time the localization and distribution of the serotonergic nerve elements in A. auriculatus was studied using immunocytochemical method and confocal laser scanning microscopy. The study revealed the presence of biogenic amine, serotonin, in the central and peripheral nervous systems of A. auriculatus, that is in the neurons and neurites of the brain ganglia, brain commissure, the longitudinal nerve cords, and the connective nerve commissures. The innervation of the attachment organs, pharynx, oesophagus and distal regions of the reproductive system by the serotonergic nerve elements is observed. The distribution of serotonergic neurons in A. auriculatus is schematically marked. The comparative analysis of findings obtained in A. auriculatus with those recorded for other digeneans reveals the presence of both conservative and distinctive features in the organization of the serotonergic nervous system in various representatives of trematodes.

Quality Assurance in Immunocytochemistry: A Review and Practical Considerations.

BACKGROUND: Cytological samples play a critical role in diagnosing advanced-stage tumors and those arising in difficult-to-reach anatomical sites such as the pancreatobiliary tract, lung, thyroid, suprarenal, pelvis, and others such as salivary glands. These samples are often the only available material for accurate diagnosis and for performing ancillary studies, such as immunocytochemistry (ICC) or the detection of molecular biomarkers. SUMMARY: While the use of immunohistochemistry is well established and standardized on formalin-fixed-paraffin-embedded histological tissue, in cytological samples, it presents unique challenges. Methods used for obtaining and processing these specimens are complex and are not standardized among laboratories. Moreover, there is also diversity in the types of cytological samples potentially suitable for ICC. KEY MESSAGES: This review explores the current landscape of ICC practices in European and North American laboratories, highlighting variability in methods and the need for standardization to ensure reliable results and reproducibility of ICC on cytological specimens.

Dual Malignancies Discovered: A Rare Case of Malignant Peritoneal Epithelioid Mesothelioma and Lung Adenocarcinoma.

Clinicians diagnosing malignant peritoneal epithelioid mesothelioma (MPM or MPeM) have historically had challenges due to the low incidence of the disease, as well as the often vague symptomatology that patients present with. Newer advances in technology, specifically in immunocytochemistry, have provided a clearer path to diagnosis. Additionally, malignant mesotheliomas must be differentiated from carcinomas. This is done via histology, immunocytochemistry, as well as a careful incorporation of the patient's clinical history. In this case, we present an asymptomatic 73-year-old non-smoker female with no past medical history of asbestos exposure. She was diagnosed with MPM following a routine abdominal hernia repair. Subsequent workup revealed a lung infiltrate that was successfully biopsied and resected, evidently found to be adenocarcinoma. A careful review of the resulting pathology, as well as the interpretation of immunocytochemistry, supported the notion that the patient had two independent malignant processes occurring at once. This case underscores the rarity of two similar, yet distinct cancers, as well as epidemiology, symptomatology, histology, immunocytochemistry, and prognosis.

The impact of different fixatives on immunostaining of lung adenocarcinomas in pleural effusion cell blocks.

BACKGROUND: Cell blocks (CBs) are widely used for biomarker analyses such as immunostaining. Although immunohistochemistry on formalin-fixed paraffin-embedded tissues is standardized, there are multiple preparation methods and fixatives for cytology. Our objective was to investigate the effect of different common fixatives on the immunoreactivity of pleural effusion CBs with metastatic lung adenocarcinomas. METHODS: This prospective study included 24 malignant pleural effusions from different patients with lung adenocarcinoma. From each case, four identical CBs were fixed in 10% neutral buffered formalin, PreservCyt, CytoLyt, and CytoRich Red (only 17 of the cases), respectively. Samples containing <100 malignant cells were excluded. All CBs were stained with thyroid transcription factor 1 (TTF-1; clones 8G7G3/1 and SPT24), napsin A, claudin 4, CEA, CK7, and epithelial cell adhesion molecule (EpCAM; clones BS14, Ber-Ep4, and MOC-31). The fraction and intensity of stained cells were evaluated. RESULTS: Of the investigated markers, a significant difference in staining proportion was seen for TTF-1 clone 8G7G3/1 and EpCAM clone MOC-31, especially with cases being negative in CytoLyt (33.3% and 83.3% positive, respectively) and PreservCyt (62.5% and 83.3%) whereas being positive in CytoRich Red (76.5% and 94.1%) and formalin (both 95.8%). A significantly weaker intensity of staining was seen for all alcohol-based fixatives compared to formalin for TTF-1 clone 8G7G3/1, napsin A, and EpCAM clone MOC-31, whereas EpCAM clone Ber-Ep4 was significantly weaker only in PreservCyt compared with formalin. CONCLUSIONS: Immunocytochemical expression and concordance with formalin-fixed CBs differ depending on the used fixative as well as the antibody and clone, warranting investigation of the reliability of each biomarker for non-formalin-fixed cytology.

p53 and Vimentin Double Immunostaining to Differentiate between High-Grade Urothelial Carcinoma Cells and Benign Atypical Cells.

INTRODUCTION: Urine cytology is an indispensable test for detecting high-grade urothelial carcinoma (HGUC); however, the distinction between HGUC cells and morphologically similar benign atypical cells poses clinical challenges. In this study, we performed double immunostaining for p53 and vimentin to establish a diagnostic method to accurately distinguish HGUC cells from benign atypical cells. METHODS: This study included 41 cases of HGUC, 11 of urolithiasis, and 22 of glomerular disease diagnosed histopathologically or clinically. After preparing urine cytology specimens from voided urine samples, p53 immunostaining was performed, and the p53-positive intensity and p53 positivity rate were calculated. Subsequently, vimentin immunostaining was performed on the same specimens to calculate the rate of vimentin positivity. RESULTS: The HGUC cell group had a mean p53-positive intensity of 2.40, a mean p53 positivity rate of 73.2%, and a mean vimentin positivity rate of 5.1%. In contrast, the mean p53-positive intensity, p53 positivity rate, and vimentin positivity rate were 1.63, 36.7%, and 66.2%, respectively, in the benign atypical cell group. There were significant differences between the two groups for each parameter. Moreover, two multiple logistic regression models combining the results of these three parameters exhibited higher sensitivity and specificity than solely assessing the p53-positive intensity, positivity rate, and vimentin positivity rate. CONCLUSION: Since double immunostaining with p53 and vimentin distinguishes HGUC cells from benign atypical cells, it could be to improve the diagnostic accuracy of urine cytology.

Extracranial metastasis from a frontal embryonal tumor to the parotid: Cytomorphologic features of a rare occurrence.

Embryonal tumors of the central nervous system (CNS) are rare and aggressive malignancies accounting for less than 1% of all central nervous system tumors. The occurrence of metastasis to extracranial sites, especially the parotid region, is highly uncommon. We present a rare case of metastatic frontal embryonal tumor (ET) in the parotid region. A 9-year-old boy presented with a progressively enlarging left parotid mass. Past history revealed that he was a known case of a frontal lobe embryonal tumor. Fine-needle aspiration cytology (FNAC) combined with immunocytochemistry from the parotid revealed a metastatic embryonal tumor. This case report highlights the importance of considering metastatic tumors in evaluating parotid masses, even in pediatric patients, and emphasizes the diagnostic potential of FNAC in diagnosing such rare and unusual tumors for prompt and appropriate patient management.