Paraffin Tissue Research Articles

From sampling to cellblock: The fully automated journey of cytological specimens.

In recent years, technological innovation have emerged to standardize pathology laboratory processes and reduce the handling of diagnostic samples. Among them is an automatic tissue embedding system that eliminates the need for manual activity in tissue paraffin embedding, thereby improving sample preservation. Unfortunately, this system cannot be used for cytological specimens due to the lack of an effective holder to support the procedure steps. In this study, we evaluated the performance of a commercial polymer matrix to enable and standardize the automatic paraffin embedding of cytological material from different organs and sources. Cytological samples from 40 patients were collected on the matrices and submitted for fully automatic workflow preparation, from formalin fixation until paraffin block, using the Sakura embedding system. Our results demonstrated the feasibility of the automated procedure, from loading cytological sample onto the matrix to obtaining the paraffin cellblock, thereby avoiding manual manipulation of cellular material. All samples resulted adequately processed and paraffin-embedded showing satisfactory tissue permeation by processing reagents, optimal preservation of cytoplasmic and nuclear details, and good quality of staining results on paraffin sections. Automated embedding of cytological samples eliminates the risk of lost specimens, reduces laboratory burden, standardizes procedures, increases diagnostic yield, and ultimately improves patients' management.

JAML overexpressed in colorectal cancer promotes tumour proliferation by activating the PI3K-AKT-mTOR signalling pathway

The expression and biological function of junctional adhesion molecule-like protein (JAML) in colorectal cancer (CRC) remain unclear. Paraffin tissue samples from 50 cases of CRC were collected to determine the expression of JAML. JAML was overexpressed or knock-down in CRC cells to evaluated the proliferation, migration and invasion in vitro and in vivo. Western-blot and others were applied to explore the mechanisms. The study showed that JAML was highly expressed within cancer tissues in 50% (25/50) of patients with CRC, and was correlated with higher TNM stage (p < 0.05). Patients of JAML-high group had poorer overall survival compared to JAML-low group (p = 0.0362, HR = 0.4295, 95% CI of 0.1908–0.9667). The tumour infiltrating lymphocytes (TILs) was lower in the JAML-high group than in the JAML-low group (p < 0.05). Overexpression of JAML promoted the proliferation, migration, and invasion of CRC by activating the PI3K-AKT-mTOR signalling pathway both in vitro and in vivo. TILs were reduced in JAML-high tumour tissues by decreasing chemokines such as CCL20 and CXCL9/10/11. Our study identified JAML, a potentially ideal target that is specifically highly expressed in CRC tissues, which promoted tumour proliferation, impaired T-lymphocytes infiltration, provided a promising therapeutic strategy for patients with CRC.

The Association of Neuroendocrine Differentiation with MicroRNA 21 and MicroRNA let7f Expression and the Clinicopathological Parameters in Primary Invasive Breast Carcinomas with Neuroendocrine Features.

MiRNAs have been reported as biomarkers with diagnostic, prognostic, and predictive value for many different diseases. Therapeutic agents targeting some miRNAs are currently being developed. We aimed to compare BC-NEFs (carcinoma of the breast with neuroendocrine features) with IDC (invasive ductal carcinoma) cases without neuroendocrine features in terms of the level of miRNA expression known to show the oncogenic (miR-21) and tumor-suppressor effects (miR-let7f) and the clinicopathological features. A total of 29 patients with a diagnosis of BC-NEFs (15 cases with neuroendocrine differentiation >50% of the whole section of tumor and 14 cases with neuroendocrine differentiation 10-50% of the tumor) and 30 patients with a diagnosis of IDC (no neuroendocrine differentiation) were retrospectively re-evaluated. Expression levels of miR-21 and miR-let7f were determined by the qRT-PCR method in paraffin tissue blocks. MiR-21 expression was significantly higher in the IDC group than in the group with BC-NEFs. miR-let7f expression was significantly lower in the group with BC-NEFs compared to the IDC group. A high expression level of miR-21 was found to be associated with progesterone receptor (PR) negativity. Our findings show that the presence of NEFs in breast carcinomas makes a significant difference in the expression levels of the investigated oncogenic (miR-21) and tumor-suppressor (miR-let7f) miRNAs. These findings suggest that miRNAs may be a potential biomarker in BC-NEFs and would benefit from targeted therapy.

Testicular mosaicism in non-mosaic postpubertal Klinefelter patients with focal spermatogenesis and in non-mosaic prepubertal Klinefelter boys.

Do testis-specific cells have a normal karyotype in non-mosaic postpubertal Klinefelter syndrome (KS) patients with focal spermatogenesis and in non-mosaic prepubertal KS boys? Spermatogonia have a 46, XY karyotype, and Sertoli cells surrounding these spermatogonia in postpubertal patients also have a 46, XY karyotype, whereas, in prepubertal KS boys, Sertoli cells surrounding the spermatogonia still have a 47, XXY karyotype. A significant proportion of patients with non-mosaic KS can have children by using assisted reproductive techniques thanks to focal spermatogenesis. However, the karyotype of the cells that are able to support focal spermatogenesis has not been revealed. Testicular biopsy samples from non-mosaic KS patients were included in the study. Karyotyping for sex chromosomes in testis-specific cells was performed by immunohistochemical analysis of inactive X (Xi) chromosome and/or fluorescent in situ hybridization (FISH) analysis of chromosomes 18, X, and Y. A total of 22 KS patients (17 postpubertal and 5 prepubertal) who were non-mosaic according to lymphocyte karyotype analysis, were included in the study. After tissue processing, paraffin embedding, and sectioning, the following primary antibodies were used for cell-specific analysis and Xi detection; one section was stained with MAGE A4 for spermatogonia, SOX9 for Sertoli cells, and H3K27me3 for Xi; the other one was stained with CYP17A1 for Leydig cells, ACTA2 for peritubular myoid cells, and H3K27me3 for Xi. Xi negative (Xi-) somatic cells (i.e. Sertoli cells, Leydig cells, and peritubular myoid cells) were evaluated as having the 46, XY karyotype; Xi positive (Xi+) somatic cells were evaluated as having the 47, XXY. FISH stain for chromosomes 18, X, and Y was performed on the same sections to investigate the karyotype of spermatogonia and to validate the immunohistochemistry results for somatic cells. According to our data, all spermatogonia in both postpubertal and prepubertal non-mosaic KS patients seem to have 46, XY karyotype. However, while the Sertoli cells surrounding spermatogonia in postpubertal samples also had a 46, XY karyotype, the Sertoli cells surrounding spermatogonia in prepubertal samples had a 47, XXY karyotype. In addition, while the Sertoli cells in some of the Sertoli cell-only tubules had 46, XY karyotype, the Sertoli cells in some of the other Sertoli cell-only tubules had 47, XXY karyotype in postpubertal samples. In contrast to the postpubertal samples, Sertoli cells in all tubules in the prepubertal samples had the 47, XXY karyotype. Our data also suggest that germ cells lose the extra X chromosome during embryonic, fetal, or neonatal life, while Sertoli cells lose it around puberty. Peritubular myoid cells and Leydig cells may also be mosaic in both postpubertal patients and prepubertal boys, but it requires further investigation. The number of prepubertal testicle samples containing spermatogonia is limited, so more samples are needed for a definitive conclusion. The fact that not all the cell nuclei coincide with the section plane limits the accurate detection of X chromosomes by immunohistochemistry and FISH in some cells. To overcome this limitation, X chromosome analysis could be performed by different techniques on intact cells isolated from fresh tissue. Additionally, there is no evidence that X chromosome inactivation reoccurs after activation of the Xi during germ cell migration during embryogenesis, limiting the prediction of X chromosome content in germ cells by H3K27me3. Our findings will lay the groundwork for new clinically important studies on exactly when and by which mechanism an extra X chromosome is lost in spermatogonia and Sertoli cells. This study was funded by The Scientific and Technological Research Council of Türkiye (TUBITAK) (2219 - International Postdoctoral Research Fellowship Program for Turkish Citizens) and the Strategic Research Program (SRP89) from the Vrije Universiteit Brussel. The authors declare no competing interests. N/A.

Genetic Characteristics of Echinococcus granulosus from Fixed Paraffin-Embedded Tissue Samples in Human Isolates Based on the High-Resolution Melting Point Analysis in Sabzevar, Northeast Iran.

There are ten genotypes of Echinococcus granulosus with different intermediate and final hosts affecting the parasite's life cycle and its transmission to humans. Therefore, this study was conducted to determine the genotype of isolated hydatid cysts using the simple and fast high-resolution melting point analysis (HRM) method. The paraffin tissue samples of patients who underwent surgery were obtained from the pathology sample bank of Vasei and Emdad Hospitals in Sabzevar, Iran during 2010-2020. The DNA content of the samples was extracted after collecting and determining the characteristics using the DNA extraction kit. PCR was performed on the samples and the presence of the hydatid cyst genome was confirmed using the special Master Kit. Mix PCR of Solis Biodyne Company and Real-Time device (Bio-Rad) were used, and the genetic identity of hydatid cysts were determined. Out of 33 paraffin samples, 21 samples contained hydatid cyst DNA, two of which were from the brain and 19 from the liver tissues; 12 samples did not contain hydatid cyst DNAs. All liver samples were from sheep species (G1), and the brain samples were from buffalo species (G3). Therefore, 9.53% of the Echinococcus species collected were buffalo (G3), and 90.47% were sheep (G1) strain. Based on previous patterns, HRM methods can be used for easy and quick identification of Echinococcus strains. The G1 strain was the dominant strain causing hydatid cyst in different human organs, including the liver and brain.

Preparation and Analysis of Histological Slides of Rat and Mouse Eyeballs to Evaluate the Retina.

A rodent eyeball is a powerful tool for researching the pathomechanisms of many ophthalmic diseases, such as glaucoma, hypertensive retinopathy, and many more. Preclinical experiments enable researchers to examine the efficacy of novel drugs, develop new methods of treatment, or seek new pathomechanisms involved in the disease's onset or progression. A histological examination provides a lot of information necessary to assess the effects of the conducted experiments and can reveal degeneration, tissue remodeling, infiltration, and many other pathologies. In clinical research, there is rarely any chance of obtaining eye tissue suitable for a histological examination, which is why researchers should take advantage of the opportunity offered by the examination of eyeballs from rodents. This manuscript presents a protocol for the histological preparation of rodent eyeballs' sections. The procedure is presented for the eyeballs of mice and rats and has the following steps: (i) harvesting the eyeball, (ii) preserving the eyeball for further analysis, (iii) processing the tissue in paraffin, (iv) preparing slides, (v) staining with hematoxylin and eosin, (vi) assessing the tissue under a light microscope. With the proposed method, the retina can be easily visualized and assessed in detail.

Challenges in Diagnosis of Melanoma from the Perspective of a Pathologist

Introduction: Melanoma is an aggressive melanocytic neoplasm with a continuously increasing incidence worldwide. Vast majority of melanoma arises from skin whereas a small subset of melanoma can arise as a primary malignancy of mucosal membranes. Gastrointestinal (GI) melanoma is usually metastatic in origin whereas primary GI melanomas are extremely rare. Anorectal Melanoma comprises of 1/% of all malignant neoplasms of this area. About 70% of these lesions are pigmented whereas 30% of them are amelanotic. Upto 25% of the patients with melanoma develops recurrent disease locally or in regional lymph nodes. Materials And Methods: This is a prospective study conducted in a tertiary care hospital comprising of six cases of Melanoma. We studied the cases according to their age, sex, clinical presentation, site, radiological findings, histopathological findings, immunohistochemical findings and treatment provided. Results: This study comprises of six cases of Melanoma among which five cases are primary one case is recurrent. Among the five primary cases, four cases have GI origin (sigmoid colon, rectum, anal canal) one case originated from skin involving left foot. There is a single case of recurrent MM involving right inguinal lymph nodes with a history of primary MM in the right lower limb. The patients belong to the age group of 6th to 8th decade with male preponderance. Histopathological examination of formalin fixed paraffin tissue sections was done followed by Hematoxylin &amp; Eosin and Immunohistochemical staining which confirmed the diagnosis of Melanoma. Conclusion: Melanoma is an aggressive melanocytic neoplasm that carries a poor prognosis. It commonly involves skin. Its occurrence in GI tract is rare in which primary lesions are exceedingly unusual. Diagnosis and Treatment require multidisciplinary team approach with surgery remaining as a cornerstone of treatment.

Next-generation sequencing has diagnostic utility in challenging small/flat urothelial lesions

Small/flat urothelial lesions are challenging and currently available ancillary immunohistochemistry testing often cannot reliably distinguish between reactive lesions and urothelial carcinoma (UCa). UCa has a characteristic molecular profile, but small/flat urothelial lesions are typically considered too small to perform next generation sequencing (NGS). Herein, we present our institution's experience with utilizing comprehensive DNA-based NGS to evaluate small/flat urothelial lesions (n = 13 cases). NGS was ordered on 7/13 small/flat urothelial lesions initially diagnosed as urothelial atypia, ordered by the pathologist to aid in further diagnosis; the remaining 6/13 cases were diagnosed as urothelial carcinoma in situ (uCIS), ordered by a treating oncologist. The test was considered as adding value if it yielded pathogenic or likely pathogenic alterations previously associated with urothelial carcinoma in the literature. Macroscopic dissection was determined necessary in all cases and obtained either by scraping (7), punch biopsy (5) or scooping (1) of paraffin tissue blocks. In 4/13 cases, tumor content was considered low (<25%); in 2/13 cases, DNA quantity yield was considered below optimal (<250 ng); all cases met required DNA quantity for testing (>50 ng). Mean target coverage ranged: 498 to 985 (optimal >500 reads). NGS testing identified mutations compatible with urothelial carcinoma in all 7 cases initially diagnosed as atypical; and in one case, the tumor recurred as a lung metastasis. All 6 uCIS had NGS testing results concordant with UCa. In conclusion, despite small sample quantity with low tumor content and DNA concentration yield, NGS testing with appropriate methodology can be considered in the setting of small/flat urothelial lesions to aid in diagnosis or per oncologist request and yield interpretable results.

Gizzard Erosion and Ulceration Syndrome in Moroccan Poultry Flocks and Molecular Characterization of Fowl Adenoviruses (FAdV).

Gizzard erosion and ulceration syndrome (GEUS) is caused by a fowl adenovirus serotype 1 (FAdV-1) and was first reported in laying hens in Japan in 1993. This syndrome has emerged as an epizootic in Morocco since 2014, causing significant economic losses for the poultry industry, but no involvement of a FAdV has been confirmed. Thus, the objective of this work was to assess GEUS cases that occurred in the country and to determine the role of FAdVs in their occurrence. Investigations were based on a retrospective reassessment of tissue sections and paraffin blocks of gizzards and livers from GEUS cases between 2014 and 2021 coupled with a prospective search of cases in 2022. Gizzards and livers were fixed in 10% neutral buffered formalin for histopathologic examinations according to standard methods and stored at -20 C for molecular analysis. After deparaffinizing, 10-µm-thick tissue sections along with fresh organs were subjected to DNA extraction using a commercial kit. A primer pair specific for the Hexon gene of FAdVs was used in conventional PCR; in contrast, for real-time PCR, a primer pair targeting the 52K gene was employed. In total, 24 flock cases with characteristic GEUS were assessed between 2014 and 2022. They were nine broiler cases aged between 11 and 39 days, 11 layer cases with an age between 17 and 29 wk, two cases in meat-type breeders aged 10 and 27 wk, and two flock cases of turkey poults aged 22 and 23 days. In most cases, microscopic lesions were consistent with an ulcerative and lymphoplasmocytic ventriculitis, and pathognomonic viral intranuclear inclusion bodies within degenerate epithelial cells were identified in four broiler flock cases, four layer cases, and one case in breeders and hence were highly suggestive of a FAdV infection. Among these nine cases that were positive at the histopathologic examination, six cases were found to be FAdV-PCR positive; another four cases were negative to histology but FAdV-PCR positive. Furthermore, a sequencing analysis was conducted, providing the initial evidence of the implication of FAdV-1 from species A as the cause of GEUS in Moroccan poultry. Additionally, a phylogenetic analysis was executed to facilitate a comparison between the strains investigated in this study and those identified in diverse geographic regions and across various time periods.

Deciphering the role of non-coding RNAs SNHG18, HOXA11-AS, MEG3, and MDC1-AS1 in glioma patients: A combined experimental and computational study

BackgroundGliomas are brain tumours categorised into low-grade (I and II) and high-grade (III and IV). Unfortunately, patients with high-grade gliomas have a poor prognosis, which is why researchers are working to improve their management. One crucial area of study is the understanding of glioma tumorigenesis and progression. Recent research has shown that long non-coding RNAs (lncRNAs) play a potential role in this process. Material and methodsWe evaluated the expression of SNHG18, HOXA11-AS, MEG3, and MDC1-AS1 in 150 paraffin tissue block samples, including 37 low-grade gliomas, 58 high-grade gliomas, and 55 non-tumoural tissues. After RNA extraction and complementary DNA synthesis (cDNA), we used probe-based qPCR to evaluate the lncRNA expression level, followed by statistical analyses. ResultsThe qRT-PCR analysis revealed differential expression patterns of lncRNAs in tumour tissues compared to controls. In low-grade tumours, SNHG18 and HOXA11-AS were upregulated (SNHG18: 1.78 ± 0.21, p < 0.001; HOXA11-AS: 1.24 ± 0.60, p < 0.001), while MEG3 and MDC1-AS1 were downregulated (MEG3: 0.72 ± 0.17, p < 0.001; MDC1-AS1: 0.24 ± 0.18, p < 0.001). In high-grade tumours, SNHG18 and HOXA11-AS were further upregulated (SNHG18: 3.16 ± 0.88, p < 0.001; HOXA11-AS: 3.83 ± 0.82, p < 0.001), and MEG3 and MDC1-AS1 remained downregulated (MEG3: 0.49 ± 0.29, p < 0.001; MDC1-AS1: 0.15 ± 0.09, p < 0.001). The bioinformatic-based study conveyed that the alteration in lncRNAs expression leads to dysregulation of RNA Polymerase II, which is observed in glioblastoma. ConclusionOur results suggest that SNHG18 and HOXA11-AS (up-regulated) may act as oncogenes in high-grade gliomas. In contrast, the downregulation of MEG3 and MDC1-AS1 in high-grade gliomas could be related to tumour suppressor properties. Therefore, it could be assumed that the expression levels of these lncRNAs may serve as potential biomarkers for diagnostic and therapeutic purposes.

P-075 A new clinically relevant classification of seminiferous tubules in Klinefelter Syndrome patients based on the karyotype of the Sertoli cells

Abstract Study question Can a new seminiferous tubule classification be made that indicates the chance of sperm retrieval by TESE in Klinefelter Syndrome (KS) patients? Summary answer Spermatogonia are only present in tubules with euploid Sertoli cells. Classification of tubules based on Sertoli cell ploidy may predict sperm retrieval success with TESE. What is known already KS patients can have children thanks to focal spermatogenesis. However, this can only happen if sperm is produced. Histological classification of seminiferous tubules in these patients has been made based on the maturity of the Sertoli cells (type A and type B seminiferous tubules). However, so far there is no classification predicting the presence of germ cells. In case two X chromosomes are present, one is inactivated. The histone-3-lysine-27-tri-methylation (H3K27me3) protein is involved in X chromosome inactivation and its immunohistochemical staining may be used for a new seminiferous tubule classification based on the karyotype of Sertoli cells. Study design, size, duration Inactive X (Xi) chromosomes were immunohistochemically stained with H3K27me3 and seminiferous tubules were classified as Xi+ tubules (presence of inactive X in Sertoli cells) or Xi- tubules (absence of inactive X in Sertoli cells). Testicular biopsies were used from 17 non-mosaic KS adult patients. Participants/materials, setting, methods After tissue fixation, processing, and paraffin embedding, sections were cut at 5 µm. Immunohistochemical staining was applied with antibodies H3K27me3 (for inactive X chromosome), MAGE-A4 (for spermatogonia), and SOX9 (for Sertoli cells). Seminiferous tubules in testicular sections were classified as Xi+ or Xi- tubules and analyzed for the presence of spermatogonia or focal spermatogenesis. X chromosome number in spermatogonia and Sertoli cells was confirmed by the FISH method using chromosome 18, Y, and X probes. Main results and the role of chance A total of 311 (18±16) seminiferous tubule sections were analyzed in testicular tissue of 17 KS men. Of the 311 tubules analyzed, 253 (14±16) of the tubules were Xi- tubules and 58 (3±4) were Xi+ tubules. Spermatogonia were present in 41 of the Xi- tubules. As an exception, only one patient had spermatogonia in two Xi+ tubules, but these tubules were immature and spermatogenesis was not observed. Ongoing spermatogenesis was observed in two of Xi- tubules in one of the patients. Sixteen of the patients had both Xi- tubules and Xi+ tubules in their testicular sections. In 13 of these 16 patients with Xi- tubules, sperm was either obtained as a result of TESE or spermatogonia were observed in the Xi- tubules. Limitations, reasons for caution In Klinefelter patients, there are few seminiferous tubules due to widespread testicular fibrosis and degeneration of seminiferous tubules. Studies including more patient samples are needed for more definitive and comprehensive conclusions. Wider implications of the findings Spermatogonia are only present in tubules with euploid Sertoli cells. Therefore, we hypothesize that focal spermatogenesis only can occur in tubules with euploid Sertoli cells. Xi+ and Xi- seminiferous tubule classification based on H3K27me3 staining may thus be used to predict sperm production in KS patients. Trial registration number Not applicable

Combining non-invasive liquid biopsy and a methylation analysis to assess surgical risk for early esophageal cancer.

While the widespread use of endoscopic submucosal dissection (ESD) has significantly reduced the incidence of early esophageal cancer (ESCA), the limited ability of ESD to strip deep infiltrating esophageal lesions results in a considerable risk of intraoperative perforation. Circulating-free DNA (cfDNA) is widely used in modern tumor screening due to its non-invasive detection capabilities. A methylation analysis offers vital insights into the condition and advancement of malignancies due to its unique positioning, such as a marker of cancer. This study investigated the potential of combining a non-invasive liquid biopsy technique, along with a methylation analysis, to assess the surgical perforation risk of ESCA patients. In this study, we conducted an analysis of gene expression differences between stage I esophageal squamous carcinoma samples and healthy tissue samples using data from The Cancer Genome Atlas (TCGA) database. We also identified the genes associated with progression-free survival (PFS) in esophageal squamous carcinoma. Integrating the framework of the methylation analysis, we explored the methylated sites of these distinct genes. To refine this process, we used the Shiny Methylation Analysis Resource Tool (SMART) to conduct a comprehensive analysis of these sites. We then confirmed the stability of the methylation sites in different lesion conditions using methylation-specific quantitative polymerase chain reaction (MS-qPCR) with paraffin tissue samples collected after ESD. We analyzed RNA-sequencing data from 42 early stage ESCA patients and 17 controls, identifying 1,263 up-regulated and 460 down-regulated genes. Functional analyses revealed involvement in key pathways such as cell cycle regulation and immune responses. Furthermore, we identified 38 differentially expressed genes associated with PFS. Using SMART analysis, we found 217 hyper-methylated regions in 38 genes, suggesting potential early markers for ESCA. Validation experiments confirmed the reliability of 29 hyper-methylated regions in FFPE tissue samples and 6 regions in cfDNA. A LunaCAM model showed high accuracy [area under the curve (AUC) =0.89] in discriminating early ESCA. Integrated assessment of six highly methylated regions significantly improved predictive performance, with 90.56% sensitivity, highlighting the importance of combinatorial biomarker evaluation for early cancer detection. This study established a novel approach that integrates non-invasive testing with a methylation analysis to assess the surgical risk of early ESCA patients. The significance of changes in methylation sites in relation to lesion status should not be underestimated, as they have the potential to offer vital insights for proactive risk assessments before surgery.

Immune profiling of mouse lung adenocarcinoma paraffin tissues using multiplex immunofluorescence panel: a pilot study

BackgroundImmune profiling has become an important tool for identifying predictive, prognostic and response biomarkers for immune checkpoint inhibitors from tumor microenvironment (TME). We aimed to build a multiplex immunofluorescence (mIF) panel to apply to formalin-fixed and paraffin-embedded tissues in mice tumors and to explore the programmed cell death protein 1/ programmed cell death 1 ligand 1 (PD-1/PD-L1) axis.ResultsAn automated eight-color mIF panel was evaluated to study the TME using seven antibodies, including cytokeratin 19, CD3e, CD8a, CD4, PD-1, PD-L1, F4-80 and DAPI, then was applied in six mice lung adenocarcinoma samples. Cell phenotypes were quantified by software to explore the co-localization and spatial distribution between immune cells within the TME. This mice panel was successfully optimized and applied to a small cohort of mice lung adenocarcinoma cases. Image analysis showed a sparse degree of immune cell expression pattern in this cohort. From the spatial analysis we found that T cells and macrophages expressing PD-L1 were close to the malignant cells and other immune cells.ConclusionsComprehensive immune profiling using mIF in translational studies improves our ability to correlate the PD-1/PD-L1 axis and spatial distribution of lymphocytes and macrophages in mouse lung cancer cells to provide new cues for immunotherapy, that can be translated to human tumors for cancer intervention.

Optimal melanin removal methods for HE staining, immunohistochemistry and molecular detection

Objective: To seek the optimal melanin-removal method for hematoxylin and eosin (HE) staining, immunohistochemistry and molecular detection. Methods: Thirty-eight paraffin tissue samples of malignant melanoma diagnosed at the Fujian Cancer Hospital, Fuzhou, China between January 2018 and March 2022 were collected and used to make a tissue microarray. Melanin in these cases was removed using warm hydrogen peroxide, double oxidation depigmentation, modified potassium permanganate-oxalic acid or trichloroisocyanuric acid, followed by HE staining. The cases were divided into two cohorts: one was subject to the one of the above four methods to remove melanin first, followed by immunohistochemistry (SOX-10, Ki-67, HMB45 and Melan A), while the other was subject to immunohistochemical staining first and then a melanin removal. Following that, seventeen melanin-rich paraffin tissue samples were collected and depigmented using the methods described above. DNA extraction was then done, followed by assessments of DNA content and quality. Moreover, the completeness of melanin removal, the effect on HE and immunohistochemical staining, and the quality of DNA were compared between the depigmented methods. Results: Regarding the effectiveness of melanin removal, the modified potassium permanganate-oxalic acid and the warm hydrogen peroxide methods were the most effective, and both showed residual melanin in only 5.26% (2/38) of the cases. The trichloroisocyanuric acid method showed residual melanin in 10.53% (4/38) of the cases. The worst was the double oxidation depigmentation method, which showed pigment residue in 15.79% (6/38) of the cases. For HE staining, the percentage of good staining with the warm hydrogen peroxide method was 92.11%, higher than the other three methods. For immunohistochemical staining, the mean staining scores of immunohistochemistry first followed by melanin removal with modified potassium permanganate-oxalic acid, double oxidation and trichloroisocyanuric acid were 20.84, 26.63 and 35.02, respectively. These immunohistochemical staining scores were higher than those of melanin removal first followed by immunohistochemistry (8.70, 15.41 and 21.22, respectively). The mean staining score of melanin removal by warm hydrogen peroxide method followed by immunohistochemistry was 33.57, superior to that of immunohistochemistry followed by the melanin removal (19.96). Moreover, the staining scores of HMB45, MelanA and Ki-67 with immunohistochemical staining followed by trichloroisocyanuric acid method were 36.45, 33.79, and 36.24, respectively, while the staining score of SOX10 with melanin removal by warm hydrogen peroxide followed by immunohistochemistry was 34.39. The DNA was significantly degraded by modified potassium permanganate-oxalic acid, double oxidation depigmentation and trichloroisocyanuric acid, whereas the mean concentration of DNA extracted after melanin removal by hydrogen peroxide method was 59.59 μg/L, substantially higher than that of DNA extracted without melanin removal (30.3 μg/L, P=0.001). The A260/A280 of DNA extracted after melanin removal by hydrogen peroxide was between 1.8 and 2.0 in all cases, and the A260/A230 was above 2.0 in sixteen cases, suggesting high purity of DNA. However, the DNA extracted without removing the melanin showed poor purity, with A260/A280 below 1.8 in eight cases and A260/A230 below 2.0 in sixteen cases. Conclusions: Warm hydrogen peroxide showed the least melanin residue, superior HE staining and a minimal effect on DNA purity/quality compared to the other three methods. It thus appears most suitable for PCR, NGS and other molecular detection. Melanin removal with trichloroisocyanuric acid after immunohistochemical staining has the least melanin residual, and thus could be the most convenient and efficient. However, it is noted that the efficacy of the same depigmentation method varies with different antibodies. Therefore, the optimal depigmentation method should be selected based on the specific markers of interest.

The biodistribution of polystyrene nanoparticles administered intravenously in the chicken embryo

Nanoplastics can cause severe malformations in chicken embryos. To improve our understanding of the toxicity of nanoplastics to embryos, we have studied their biodistribution in living chicken embryos. We injected the embryos in the vitelline vein at stages 18–19. We injected polystyrene nanoparticles (PS-NPs) tagged with europium- or fluorescence. Their biodistribution was tracked using inductively-coupled plasma mass spectrometry on tissue lysates, paraffin histology, and vibratome sections analysed by machine learning algorithms. PS-NPs were found at high levels in the heart, liver and kidneys. Furthermore, PS-NPs crossed the endocardium of the heart at sites of epithelial-mesenchymal transformation; they also crossed the liver endothelium. Finally, we detected PS-NPs in the allantoic fluid, consistent with their being excreted by the kidneys. Our study shows the power of the chicken embryo model for analysing the biodistribution of nanoplastics in embryos. Such experiments are difficult or impossible in mammalian embryos. These findings are a major advance in our understanding of the biodistribution and tissue-specific accumulation of PS-NPs in developing animals.

Determination of local expressions of IGF-1, LC3B and NF-kB in white muscle disease in lambs by immunohistochemical method

White muscle disease (WMD) is also known as Stiff Lamb Disease or Nutritional Muscular Dystrophy. Selenium and/or Vitamin E deficiency constitutes the etiology of the disease. This study aimed to immunohistochemically evaluate local protein expressions of Nuclear factor kappa B (NF-kB), Insulin-like growth factor-1 (IGF-1) and Microtubule-related protein 1A/1B-light chain 3 beta (LC3B) in WMD. The material of the study consisted of 15 WMD, and 6 healthy lamb heart samples. The heart tissues of the autopsied lambs were subjected to routine tissue processing and paraffin blocks were obtained. Then, it was stained with Hematoxylin-Eosin and immunohistochemical methods. Control group lambs had normal macroscopic appearance. Macroscopically, hyaline degeneration and zenker’s necrosis, calcification areas were observed in WMD tissues. Microscopically, degenerative and necrotic muscle fibers, calcification areas, fibrosis, mononuclear cell infiltrates and macrophage infiltrates were detected in WMD heart tissues. Immunohistochemically, significant increases were detected in IGF-1 (p

The role of FERMT2 in the tumor microenvironment and immunotherapy in pan-cancer using comprehensive single-cell and bulk sequencing

FERMT2 has been identified as a participant in integrin-linked kinase signaling pathways, influencing epithelial-mesenchymal transition and thereby affecting tumor initiation, progression, and invasion. While the character of FERMT2 in the tumor microenvironment (TME) as well as its implications for immunotherapy remain unclear. Thus, we conducted a comprehensive analysis to assess the prognostic significance of FERMT2 using Kaplan-Meier analysis. In addition, we employed enrichment analysis to uncover potential underlying molecular mechanisms. Using "Immunedeconv" package, we evaluated the immune characteristics of FERMT2 within TME. Furthermore, we determined the expression levels of FERMT2 in various cell types within TME, based on single-cell sequencing data. To confirm the co-expression of FERMT2 and markers of cancer-associated fibroblasts (CAFs), we performed multiplex immunofluorescence staining on tissue paraffin sections across various cancer types. Our analysis disclosed a significant correlation between elevated FERMT2 expression and unfavorable prognosis in specific cancer types. Furthermore, we identified a strong correlation between FERMT2 expression and diverse immune-related factors, including immune checkpoint molecules, immune cell infiltration, microsatellite instability (MSI), and tumor mutational burden (TMB). Additionally, there was a significant correlation between FERMT2 expression and immune-related pathways, particularly those associated with activating, migrating, and promoting the growth of fibroblasts in diverse cancer types. Interestingly, we observed consistent co-expression of FERMT2 in both malignant tumor cells and stromal cells, particularly within CAFs. Notably, our findings also indicated that FERMT2, in particular, exhibited elevated expression levels within tumor tissues and co-expressed with α-SMA in CAFs based on the multiplex immunofluorescence staining results.

Rapid examination of lung tissues by nonlinear microscopy.

Traditional histopathology is a time-intensive and labor-intensive process involving tissue formalin fixation, paraffin embedding, and microtoming into thin sections for H&E staining. Frozen section analysis is a modality used during surgery to quickly evaluate tissue, but it has limitations, such as the size and number of the specimens that can be analyzed as well as difficulties with fatty and bony tissues. Our objective was to investigate the performance of nonlinear microscopy, a fluorescence microscopy technique, for the rapid examination of resected lung tumors. In this proof-of-principle study, nonlinear microscopy imaging of resected lung tissue was performed on a total of 73 tissue specimens collected from 13 patients who underwent lobectomy, segmentectomy, or wedge resection for pulmonary nodules. Two pathologists reviewed the digital nonlinear microscopy images in comparison to the corresponding histopathologic H&E slides from a variety of pulmonary pathologies. This study demonstrated that nonlinear microscopy readily replicates traditional H&E staining for both lung tumors and nonneoplastic pulmonary structures. Nonlinear microscopy provides many advantages over frozen section analysis and is an optical imaging platform that has the potential to augment rapid pathologic evaluation of resected tissues in the age of digital pathology.

A three miRNAs panel in paraffin tissue serves as tool for predicting prognosis of renal cell carcinoma.

Renal cell carcinoma (RCC) stands as the most prevalent form of urogenital cancer. However, there is currently no universally accepted method for predicting the prognosis of RCC. MiRNA holds great potential as a prognostic biomarker for RCC. A total of 100 cases with complete paraffin specimens and over 5-year follow-up data meeting the requirements were collected. Utilizing the clinical information and follow-up data of the specimens, an information model was developed. The expression levels of eight microRNAs were identified using RT-qPCR. Finally, determine and analyze the clinical application value of these microRNAs as prognostic markers for RCC. Significant differences were observed in the expression of two types of miRNAs (miR-378a-5p, miR-23a-5p) in RCC tissue, and three types of miRNAs (miR-378a-5p, miR-642a-5p, miR-23a-5p) were found to be linked to the prognosis of RCC. Establish biomarker combinations of miR-378a-5p, miR-642a-5p, and miR-23a-5p to evaluate RCC prognosis. The combination of three microRNA groups (miR-378a-5p, miR-642a-5p, and miR-23a-5p) identified in paraffin section specimens of RCC in this study holds significant potential as biomarkers for assessing RCC prognosis.

Non-neoplastic Kidney Diseases in Adult Tumor Nephrectomy and Nephroureterectomy Specimens in a Southeast Asian Tertiary Medical Center

OBJECTIVE: Non-neoplastic kidney diseases are concurrently present in up to 15% of patients with renal and ureteral tumors, which have been most studied in the United States. This study was conducted to determine the prevalence and spectrum of renal parenchymal diseases in similar patients from a Southeast Asian academic institution.METHODS: We searched the database of the Department of Anatomical Pathology, Faculty of Medicine Vajira Hospital, Navamindradhiraj University (2012–2022) and found 194 adult nephrectomy and nephroureterectomy specimens with renal, renal pelvis, and ureteral tumors. Additional stains included periodic acid-Schiff, methenamine silver, and Masson trichrome. Direct immunofluorescence microscopy was performed on the paraffin tissue sections for immunoglobulin (Ig) G, IgA, IgM, and kappa and lambda light chains. Clinical information, including age, gender, and co-morbidities, was obtained from the electronic medical records.RESULTS: Analysis of the 194 cases demonstrated the average age was 61 years (range: 17-89 years), with 126 males (65%) and 68 females (35%). After re-examination of the non-neoplastic renal parenchyma, 14 cases (7%) had diffused and/or nodular mesangial sclerosis. Diabetic nephropathy (12 cases) and idiopathic nodular glomerulosclerosis (2 cases) were diagnosed and associated with either stage 1 or 2 genitourinary cancers. Another case was diagnosed with atheroembolic renal disease. In all cases, the concurrent renal diseases were not identified during the initial evaluation.CONCLUSION: Examination of the non-neoplastic renal parenchyma is important to identify non-neoplastic kidney diseases, such as diabetic nephropathy, so early treatment may result in improved clinical outcomes for kidney and urothelial cancer patients.