BACKGROUND: 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.
METHODS: 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).
CONCLUSIONS: 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.

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.

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.

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.

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.

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), β(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 α2β2γ2-GABAARs versus extrasynaptic type α4β3δ-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 α2β2γ2-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 α2β2γ2-GABAAR/NL2 showing a significantly greater synaptogenic activity than α4β3δ-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 α2β2γ2-GABAAR was established and mapped to the large intracellular domain of the γ2 subunit.

Cervical cancer screening is a challenge mainly in developing countries. In developed countries, both incidence and mortality rates have been decreasing due to well organized screening programs. One of the potential biomarkers being exploited are the minichromosome maintenance proteins (MCMs), which show both specificity and sensitivity. MCM2-7 are involved in DNA replication initiation and elongation, and the MCM subunits are highly expressed in malignant tissues. Unlike other MCMs, MCM10, which is not part of the core helicase complex, is a critical determinant of origin activation and its levels are limiting in cancer cells. In this study, we performed bioinformatic analysis on the expression profile of all DNA replication associated MCM proteins in cervical cancer. MCM10 showed a relatively higher expression profile compared to the other MCMs. The mRNA expression levels of the MCMs were significantly increased in tumour tissues compared to normal, and MCM10 showed a fold change of 3.4. In order to understand if MCM10 is associated with the aggressiveness of cervical cancer, we looked into the mRNA expression pattern of MCM10 in three cervical cancer cell lines and one normal cervical cell line. MCM10 expression was significantly higher in the case of the more aggressive cancer cell line HeLa compared to controls. MCM10, therefore, can serve as a prominent biomarker for cancer progression and thus aid in early detection to control the spread of cancer cells. Our results show that MCM10 expression levels in cervical cancer cell lines are associated with cancer aggressiveness, demonstrating its clinical significance.

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.

BACKGROUND: 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.
CONCLUSIONS: 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.

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.