Fixation Of Specimens Research Articles

Application of Information Link Control in Surgical Specimen Near-Miss Events in a South China Hospital: Nonrandomized Controlled Study.

Information control is a promising approach for managing surgical specimens. However, there is limited research evidence on surgical near misses. This is particularly true in the closed loop of information control for each link. A new model of surgical specimen process management is further constructed, and a safe operating room nursing practice environment is created by intercepting specimen near-miss events through information safety barriers. In a large hospital in China, 84,289 surgical specimens collected in the conventional information specimen management mode from January to December 2021 were selected as the control group, and 99,998 surgical specimens collected in the information safety barrier control surgical specimen management mode from January to December 2022 were selected as the improvement group. The incidence of near misses, the qualified rate of pathological specimen fixation, and the average time required for specimen fixation were compared under the 2 management modes. The causes of 2 groups of near misses were analyzed and the near misses of information safety barrier control surgical specimens were studied. Under the information-based safety barrier control surgical specimen management model, the incidence of adverse events in surgical specimens was reduced, the reporting of near-miss events in surgical specimens was improved by 100%, the quality control quality management of surgical specimens was effectively improved, the pass rate of surgical pathology specimen fixation was improved, and the meantime for surgical specimen fixation was shortened, with differences considered statistically significant at P<.05. Our research has developed a new mode of managing the surgical specimen process. This mode can prevent errors in approaching specimens by implementing information security barriers, thereby enhancing the quality of specimen management, ensuring the safety of medical procedures, and improving the quality of hospital services.

CYTOLOGICAL DIAGNOSTICS OF THYROID DISEASES: BASIC PRINCIPLES AND FEATURES OF APPLICATION IN MODERN CONDITIONS

Introduction. Cytological diagnostics of thyroid diseases widely used in medical practice today. The standards and guidelines used in this examination ensure close cooperation between doctors of different specialties. Aim of research: To determine the methodological and organizational features of cytological examination of the thyroid gland, which can improve the use of this diagnostic method in Ukraine. The effectiveness of cytological examination depends on many factors. First of all, it is important to use this method in cases where the test result can be sufficiently informative and help identify thyroid diseases, choose further diagnostic and appropriate treatment tactics. Important stages in the actual cytological examination of thyroid diseases are: sampling, fixation and staining of cytological specimens, evaluation of microscopic changes, formulation of a cytological conclusion, and, if necessary, the use of immunocytochemical and molecular genetic methods. Compared to other countries, Ukraine has a higher incidence of uninformative reports in cytological diagnostics of thyroid diseases. The reasons for this are most likely more frequent violations of the technique of puncture, fixation and transportation of the material. It should be noted that cytologic examination in Ukraine is performed by a laboratory doctors, while in most countries of the world this examination is performed by a pathologist. Conclusions. In order to improve the effectiveness of the method of cytological diagnostics of thyroid diseases in Ukraine, it is necessary to ensure proper compliance with the relevant norms, standards and rules at all stages of its implementation. Taking into account the current regulatory norms and rules for cytological examinations in Ukraine, it is necessary to improve the qualifications of laboratory doctors in the field of cytological diagnostics of thyroid diseases, officially to include the method of cytological diagnostics into the work of a pathologist and to actively implement in medical practice The Bethesda System for Reporting Thyroid Cytopathology, 2023.

Be bold, start cold! cold formalin fixation of colorectal cancer specimens granted superior DNA and RNA quality for downstream molecular analysis

The use of cold formalin fixation (CFF; i.e., fixating tissue samples with 4 °C precooled formalin) recently attracted further attention owing to its putative improved ability to preserve nucleic acid compared with standard room temperature formalin (SFF). In this study, we aimed to assess the effect of four formalin-based fixation protocols (SFF, CFF, delayed formalin fixation-DFF, and cold formalin hyperfixation; CFH) on both DNA and RNA quality. We collected 97 colorectal cancer (CRC) and analyzed 23 metrics of nucleic acid quantity and quality yield using a multiplatform approach by combining spectrophotometric, fluorimetric, electrophoretic, and polymerase chain reaction (PCR) assays. Following confirmation of fixation-protocol-related different effects via clustering analysis, CFF presented best metrics compared with all protocols, specifically positive coefficients of DV1000-60000, DV2/DV1, DNA λ ratio 260/230, and ABL gene expression absolute copies, and negative coefficient of DV150-1000. The SFF subgroup presented a positive coefficient of DV150-1000 and negative coefficients for DV1000-60000, DV2/DV1, RNA λ ratio 260/230, RNA QuBit concentration, DV100/200, RNA electrophoresis concentration and absolute quantity, and ABL copies. Overall, we confirmed the superior yield performances of CFF preservation for both DNA and RNA compared with the other protocols in our series of CRC samples. Pending further validations and clarification of the specific mechanisms behind these findings, our study supports the implementation of CFF in the pathology unit routine specimen management for tumor tissue molecular profiling.

Anatomical variations and embryological basis of arch of aorta and aortic valve.

Variations in the arch of the aorta and aortic valves among fetal, cadaveric, and post-mortem specimens present a spectrum of anatomical configurations, posing challenges in establishing a standard norm. While some variations hold surgical significance, many bear little functional consequence but provide insights into embryological origins. The aortic arch exhibits diverse branching patterns, including common trunks and different orders, relevant for endovascular surgeries. Meanwhile, malformations in the aortic valve, affecting the aorta, may lead to ischemia and cerebral infarction, warranting understanding of coexisting arch and valve anomalies to predict complications like aortic dissection. Studies in the Indian population mirror global variations, underscoring the need to explore embryological, clinical, and surgical implications for safer vascular surgeries involving the aortic arch and valves. The study's objectives included examining branching patterns, diameters, and distances between arch branches and exploring aortic valve variations. Employing a cross-sectional design, the study was conducted across Anatomy, Forensic Medicine, and Obstetrics and Gynecology departments. A sample of 100, comprising cadavers, fetuses, and postmortem specimens, were gathered. Specimens ranged from 14weeks of intrauterine life to 85years, with intact thoracic cages as inclusion criteria. Methodology involved dissection, specimen fixation, and macroscopic examination for variations and morphological parameters. Results showed aortic diameter increase with age, with significant gender differences. A statistically significant association between arch variations and anomalous valves was observed, suggesting mutual predictability. Individuals with valve anomalies should undergo comprehensive cardiology evaluation to avert complications like aortic dissection during endovascular surgeries. While atheromatous plaques were prevalent in younger groups, their frequency rose with age, necessitating vigilant vascular monitoring. Careful handling during surgeries is paramount, given potential adverse outcomes resulting from variations. Overall, the study underscores the importance of comprehensive anatomical understanding in clinical contexts, guiding effective management strategies and ensuring patient safety in vascular surgeries.

Mitochondrial genome data provide insights into the phylogenetic relationships within Triplophysadalaica (Kessler, 1876) (Cypriniformes, Nemacheilidae).

Due to the detrimental effect of formaldehyde on DNA, ethanol has replaced formalin as the primary preservative for animal specimens. However, short-term formalin fixation of specimens might be applied during field collection. In an increasing number of studies, DNA extraction and sequencing have been successfully conducted from formalin-fixed specimens. Here the DNA from five specimens of Triplophysadalaica (Kessler, 1876) were extracted and performed high-throughput sequencing. Four of the specimens underwent short-term fixation with formalin and were subsequently transferred to ethanol. One was continuously stored in ethanol. No significant difference of DNA quality and amount were observed among these samples. Followed by assembly and annotation, five mitochondrial genomes ranging in length from 16,569 to 16,572 bp were obtained. Additionally, previously published data of other individuals or species were included to perform phylogenetic analyses. In the reconstructed trees, all eight individuals of T.dalaica form a monophyletic group within the Triplophysa branch. The group is divided into three clades: (1) samples from the Yellow River, (2) those from the Yangtze River, and (3) those from the Haihe River, and the Lake Dali Nur. This study sheds initial light on the phylogeographic relationships among different populations of T.dalaica, and will support the research about its evolutionary history in the future.

Unravelling cryptic diversity in the Paratrichodorus allius-group species complex to resolve eight new species of the genus and new insights on the molecular phylogeny (Nematoda: Trichodoridae)

Abstract The stubby-root nematode genus Paratrichodorus comprises species of plant polyphagous ectoparasitic nematodes distributed worldwide and a few of them are plant virus-vectors (tobravirus). This is one of the most difficult nematode genera in terms of species identification, since it is phenotypically conserved with closely related species and is highly sensitive to the specimen fixation and preservation processes. Using an integrative and multidisciplinary taxonomy framework, we determined species boundaries among the Paratrichodorus populations from a P. allius species-complex sampled in cultivated and natural ecosystems across Spain. We support the hypothesis that nematode diversity of the P. allius species-complex remains underestimated and describe here a total of eight new species on the basis of detailed morphological and molecular characterization of 16 Paratrichodorus populations: Paratrichodorus asidonaensis sp. nov., Paratrichodorus benalupensis sp. nov., Paratrichodorus hinogerus sp. nov., Paratrichodorus litoralis sp. nov., Paratrichodorus paraallius sp. nov., Paratrichodorus rocianerus sp. nov., Paratrichodorus tarifaensis sp. nov., and Paratrichodorus uliaensis sp. nov. This study emphasizes the importance of properly applying a broad taxonomic framework in plant-parasitic nematodes. It is also relevant for plant health, since it correctly delimits species morphologically closed to the virus vector nematode, P. allius, and gives proper molecular markers for its unequivocal identification.

Hot alkaline lysis gDNA extraction from formalin-fixed archival tissues.

Formalin fixation of natural history specimens and histopathological material has historically been viewed as an impediment to successful genomic analysis. However, the development of extraction methods specifically tailored to contend with heavily crosslinked archival tissues, re-contextualises millions of previously overlooked specimens as viable molecular assets. Here, we present an easy-to-follow protocol for screening archival wet specimens for molecular viability and subsequent genomic DNA extraction suitable for sequencing. The protocol begins with non-destructive assessment of specimen degradation and preservation media conditions to allow both museum curators and researchers to select specimens most likely to yield an acceptable proportion (20-60%) of mappable endogenous DNA during short-read DNA sequencing. The extraction protocol uses hot alkaline lysis in buffer (0.1M NaOH, 1% SDS, pH 13) to simultaneously lyse and de-crosslink the tissue. To maximise DNA recovery, phenol:chloroform extraction is coupled with a small-fragment optimised SPRI bead clean up. Applied to well-preserved archival tissues, the protocol can yield 1-2 μg DNA per 50 mg of tissue with mean fragment sizes typically ranging from 50-150 bp, which is suitable to recover genomic DNA sufficient to reconstruct complete mitochondrial genomes and achieve up to 25X nuclear genome coverage. We provide guidance for read mapping to a reference genome and discuss the limitations of relying on small fragments for SNP genotyping and de novo genome assembly. This protocol opens the door to broader-scale genetic and phylogenetic analysis of historical specimens, contributing to a deeper understanding of evolutionary trends and adaptation in response to changing environments.

CytogeneticsTissue Fixation Significantly Impacts MLH1 Promoter Methylation Analysis: A Quality Improvement Study

Abstract MLH1 promoter methylation (MPM) analysis is an invaluable tool for distinguishing sporadic from inherited colorectal and endometrial cancers. At our institutional clinical genomics lab, we observed an unusually high failure rate of MPM testing in endometrial (EM) cancers. This triggered a quality improvement (QI) project to identify the underlying cause and improve the success rate of our MPM assay. We retrospectively identified all MPM cases (EM and non-endometrial (NEM) cancers) between January 2019 and October 2022 through querying our departmental database. The failure rates of these two groups were juxtaposed with the following variables: DNA concentration, performing technologists, operating surgeons, and specimen collection day (Monday through Saturday). A subsequent fishbone analysis was performed to evaluate the impact of specimen handling, fixation, and embedding procedure/timeline in the gross room and histology labs. We identified one-hundred thirty-one EM cases and eighty-two NEM cases that underwent MPM testing. Thirty-one EM cases (24%) and seven NEM cases (9%) failed, re-demonstrating the higher failure rate (X2=7.9, P-value=0.005) of EM cases. There was no significant difference in DNA concentration, performing technologists, and operating surgeons when compared to passed cases, however, a highly significant difference was seen concerning day of specimen collection (X2=53, P-value&amp;lt;0.0001). Most notably, all failed EM (n=31) and NEM cases (n=7) were collected on either Thursday or Friday (failure rate of 26% and 59%, respectively, 44% jointly). Our fishbone analysis revealed that specimens accessioned Thursday afternoon and Friday were fixed throughout the weekend before embedding on Monday, resulting in prolonged fixation (one extra day). This is consistent with previous studies’ conclusions showing DNA quality (but not quantity) significantly worsened with longer formalin fixation. Upon presenting these data to lab personnel, workflow changes were implemented. Cancer tissue that would have originally fixed over the weekend was instead processed on Saturday starting December 2022. This QI project revealed that prolonged formalin fixation of specimens over the weekend dramatically reduces the success rate of MPM analysis of EM cancer cases. Subsequently, we expanded our investigation to include other molecular assays at our institution that have returned similar results. Since the implementation of the revised workflow in December 2022, a preliminary prospective analysis showed the MLH1 failure rate of EM cancer tissues collected on Thursday and Friday has decreased from 44% to 11% in the past three months (December 2022 – February 2023). The current findings have important implications for the use of FFPE specimens in genetic analysis and highlight the need for careful consideration of fixation workflows, especially over the weekend, in sample processing protocols.

Glyoxal acid-free (GAF) histological fixative is a suitable alternative to formalin: results from an open-label comparative non-inferiority study

Formalin, an aqueous solution of formaldehyde, has been the gold standard for fixation of histological samples for over a century. Despite its considerable advantages, growing evidence points to objective toxicity, particularly highlighting its carcinogenicity and mutagenic effects. In 2016, the European Union proposed a ban, but a temporary permission was granted in consideration of its fundamental role in the medical-diagnostic field. In the present study, we tested an innovative fixative, glyoxal acid-free (GAF) (a glyoxal solution deprived of acids), which allows optimal tissue fixation at structural and molecular level combined with the absence of toxicity and carcinogenic activity. An open-label, non-inferiority, multicentric trial was performed comparing fixation of histological specimens with GAF fixative vs standard phosphate-buffered formalin (PBF), evaluating the morphological preservation and the diagnostic value with four binary score questions answered by both the central pathology reviewer and local center reviewers. The mean of total score in the GAF vs PBF fixative groups was 3.7 ± 0.5 vs 3.9 ± 0.3 for the central reviewer and 3.8 ± 0.5 vs 4.0 ± 0.1 for the local pathologist reviewers, respectively. In terms of median value, similar results were observed between the two fixative groups, with a median value of 4.0. Data collected indicate the non-inferiority of GAF as compared to PBF for all organs tested. The present clinical performance study, performed following the international standard for performance evaluation of in vitro diagnostic medical devices, highlights the capability of GAF to ensure both structural preservation and diagnostic value of the preparations.

Could immediate second-look endoscopy reduce post-endoscopic submucosal dissection bleeding?

Background and study aimGastric endoscopic submucosal dissection (ESD) is a curative treatment for gastric neoplasm. A scheduled second-look endoscopy could be performed to prevent delayed post-ESD bleeding. However, no studies on the efficacy of second-look endoscopy for the prevention of early delayed post-ESD bleeding have been conducted. So, the aim of this study was to be the first to investigate the efficacy of immediate second-look endoscopy for the prevention of post-ESD bleeding. Patients and methodsThe 266 gastric ESD cases were included. Immediate second-look endoscopy was defined as repeated upper endoscopy soon after complete hemostasis of the ESD site and specimen fixation. Early and late delayed bleeding were classified as before or after 24 h after the ESD, respectively. ResultsThe 262 ESD cases were enrolled and divided into three groups: the immediate second-look (n = 79), scheduled second-look (n = 86), and no second-look (n = 97). Post-ESD bleeding occurred in 19 cases (7.3%). Of these, 13 (68.4%) were early delayed post-ESD bleeding. The immediate second-look endoscopy had a lower incidence of early delayed post-ESD bleeding compared to the groups without immediate second-look endoscopy, (3.8% vs. 0.8%, p = 0.009). In a multivariate analysis, immediate second-look endoscopy significantly reduced early delayed post-ESD bleeding (OR 0.39, p = 0.022). The resected specimen area ≥ 1,000 mm2 was an independent risk factor for early delayed post-ESD bleeding (OR 8.98, p = 0.010). However, the frequency of delayed post-ESD bleeding did not differ between the three groups. ConclusionImmediate second-look endoscopy after gastric ESD may prevent early delayed post-ESD bleeding under certain circumstances.

Evaluation of A Computer-Aided Detection Software for Prostate Cancer Prediction: Excellent Diagnostic Accuracy Independent of Preanalytical Factors

Prostate cancer (PCa) is the most common noncutaneous cancer in men in the Western world. In addition to accurate diagnosis, Gleason grading and tumor volume estimates are critical for patient management. Computer-aided detection (CADe) software can be used to facilitate the diagnosis and improve the diagnostic accuracy and reporting consistency. However, preanalytical factors such as fixation and staining of prostate biopsy specimens and whole slide images (WSI) on scanners can vary significantly between pathology laboratories and may, therefore, impact the quality of WSI and utility of CADe algorithms. We evaluated the performance of a CADe software in predicting PCa on WSIs of prostate biopsy specimens and focused on whether there were any significant differences in image quality between WSIs obtained on different scanners and specimens from different histopathology laboratories. Thirty prostate biopsy specimens from 2 histopathology laboratories in the United States were included in this study. The hematoxylin and eosin slides of the biopsy specimens were scanned on 3 scanners, generating 90 WSIs. These WSIs were then analyzed using a CADe software (INIFY Prostate, Algorithm), which identified and annotated all areas suspicious for PCa and calculated the tumor volume (percentage area of the biopsy core involved). Study pathologists then reviewed the Algorithm’s annotations and tumor volume calculation to confirm the diagnosis and identify benign glands that were misclassified as cancer (false positive) and cancer glands that were misclassified as benign (false negative). The CADe software worked equally well on WSIs from all 3 scanners and from both laboratories, with similar sensitivity and specificity. The overall sensitivity was 99.4%, and specificity was 97%. The percentage of suspicious cancer areas calculated by the Algorithm was similar for all 3 scanners. For WSIs with small foci of cancer (<1 mm), the Algorithm identified all cancer glands (sensitivity, 100%). Preanalytical factors had no significant impact on whole slide imaging and subsequent application of a CADe software. Prediction accuracy could potentially be further improved by processing biopsy specimens in a centralized histology laboratory and training the Algorithm on WSIs from the same laboratory in order to minimize variations in preanalytical factors and optimize the diagnostic performance of the Algorithm.

Walther Flemming: life path and scientific legacy

The main stages of Walther Flemming life and activity are considered in the article. Walther Flemming (21.04.1843–4.08.1905) is German biologist, anatomist, one of the most famous histologists of the 19th century and the founder of cytogenetics. He studied medicine at four famous universities of the German Empire – in Göttingen, Tübingen, Berlin and Rostock. After defending his thesis, in 1868 he started teaching anatomy and histology at Würzburg, Amsterdam and Rostock universities, Germany and Charles Ferdinand University, the Czech Republic. During the last 25 years of his life, he was a professor of anatomy at the Christian Albrecht University in Kiel, Germany and headed one of its departments – the anatomical institute.&#x0D; At the beginning of W. Flemming scientific activity, his research mostly concerned histology and anatomy, as well as the development of histological methods and fixation of microscopic specimens. However, the main works of the scientist are devoted to study cell division process and the chromosomes distribution in daughter nuclei. He firstly introduced the concept of “mitosis”, which he studied on stained microscopic preparations, using salamander epithelial cells as a source of biological material. W. Flemming described the processes in the nucleus, distinguished “progressive” and “regressive” phases of cell division. He demonstrated that the cells contain areas that absorb basophilic dyes. These areas were later called chromatin; other structures remained unstained under such conditions and were therefore called achromatin. W. Flemming studied the structure and functions of the spindle during cell division, and he also studied the polar structure, which he called centriole. The scientist found that indirect division usually occurs not only in vertebrates, but also in invertebrates and protists. He established that two successive, morphologically distinct divisions of nuclear occur during spermatogenesis, resulting in the division of chromatin into two daughter cells. Thus, W. Flemming’s research contributed to the development of cell biology and its branches.

The risk of weekend biopsy: Impact of specimen source and fixation status on HER2 assessment in the treatment of advanced gastric cancer (The HER_WEEKEND study).

Accurate evaluation of human epidermal growth factor receptor type 2 (HER2) expression is crucial for determining chemotherapy regimens in gastric cancer. However, formalin fixation status has been identified as an important factor affecting HER2 assessment reliability. This retrospective cohort study aimed to investigate the correlation between sample collection day (weekday vs. weekend) and source (biopsy vs. surgical specimens) in assessing HER2 expression in patients with unresectable advanced/recurrent gastric cancer. Data were collected from gastric cancer patients who received chemotherapy at a single public hospital in Japan from 2008 to 2021. The analysis included 177 patients (109 men, 68 women) with a median age of 68.0 (21-88) years, and the primary outcome was the HER2 positivity rate. The overall HER2 positivity rate was 18.1%, with higher rates on weekdays (20.0%) compared to weekends (12.8%). Biopsies had higher positivity rates on weekdays (23.9%) but lower rates on weekends (11.1%) than surgical specimens. Significant differences were observed in formalin fixation times between weekdays and weekends for both biopsies and surgical samples. The study findings suggest that longer formalin fixation times on weekends may lead to underestimating HER2 expression, particularly in biopsies. Therefore, it is crucial to be cautious of excessive formalin fixation when collecting samples, especially during weekend biopsies.

Analysis of fixation materials in micro-CT: It doesn't always have to be styrofoam.

Good fixation of filigree specimens for micro-CT examinations is often a challenge. Movement artefacts, over-radiation or even crushing of the specimen can easily occur. Since different specimens have different requirements, we scanned, analysed and compared 19 possible fixation materials under the same conditions in the micro-CT. We focused on radiodensity, porosity and reversibility of these fixation materials. Furthermore, we have made sure that all materials are cheap and easily available. The scans were performed with a SkyScan 1173 micro-CT. All dry fixation materials tested were punched into 5 mm diameter cylinders and clamped into 0.2 ml reaction vessels. A voxel size of 5.33 μm was achieved in a 180° scan in 0.3° steps. Ideally, fixation materials should not be visible in the reconstructed image, i.e., barely binarised. Besides common micro-CT fixation materials such as styrofoam (-935 Hounsfield Units) or Basotect foam (-943 Hounsfield Units), polyethylene air cushions (-944 Hounsfield Units), Micropor foam (-926 Hounsfield Units) and polyurethane foam, (-960 Hounsfield Units to -470 Hounsfield Units) have proved to be attractive alternatives. Furthermore, more radiopaque materials such as paraffin wax granulate (-640 Hounsfield Units) and epoxy resin (-190 Hounsfield Units) are also suitable as fixation materials. These materials often can be removed in the reconstructed image by segmentation. Sample fixations in the studies of recent years are almost all limited to fixation in Parafilm, Styrofoam, or Basotect foam if the fixation type is mentioned at all. However, these are not always useful, as styrofoam, for example, dissolves in some common media such as methylsalicylate. We show that micro-CT laboratories should be equipped with various fixation materials to achieve high-level image quality.

Prediction Formula for Pathological Depth of Invasion From Clinical Depth of Invasion in Tongue Squamous Cell Carcinoma (SCC) Stage I/II Cases.

The depth of invasion (DOI) of tongue squamous cell carcinoma (SCC) is an important prognostic factor. The definition is clear for pathological DOI (pDOI), but the treatment strategy is determined by the preoperative clinical DOI (cDOI). Few studies have investigated the difference between these DOIs. The purpose of this study was to obtain the correlation equation between cDOI and pDOI for Stage I/II tongue SCC and to consider the points to be noted in actual clinical practice. In this retrospective study, 58 patients with clinical stage I/II tongue SCC were included. Correlations between cDOI and pDOI were obtained for all 58 cases, as well as for 39 cases which excluded superficial and exophytic lesions. The overall cDOI and pDOI median values were 8.0 and 5.5 mm, respectively; the 2.5 mm reduction was significant (p< 0.01). The correlation equation was pDOI = 0.81 × cDOI-0.23 (r = 0.73). Furthermore, re-analysis of the 39 cases revealed that pDOI = 0.84 × cDOI-0.37 (r = 0.62). Hence, a derived equation pDOI = 0.84 × (cDOI-0.44) was obtained to predict pDOI from cDOI. This study indicated that it is necessary to consider contraction due to specimen fixation by subtracting the thickness of the mucosal epithelium. Clinical T1 cases with a cDOI of 5 mm or less had a pDOI of 4 mm or less, and it would be expected to have low positive rate of neck lymph node metastasis.

Evaluation of resection margins in oral squamous cell carcinoma.

Surgery is the primary treatment modality for oral squamous cell carcinoma. The purpose of the surgical procedure is complete removal of the tumor with asufficient margin of healthy tissue in its surroundings. Resection margins represent an important factor for planning further treatment and for estimation of the disease prognosis. Resection margins can be divided into negative, close and positive. Positive resection margins are considered aprognostically unfavorable factor. However, the prognostic significance of close resection margins is not entirely clear. The aim of this study was to evaluate the relationship between resection margins and disease recurrence, disease-free survival and overall survival. The study included 98 patients who underwent surgery for oral squamous cell carcinoma. During histopathological examination, resection margins of each tumor were evaluated by apathologist. The margins were divided into negative (&gt; 5 mm), close (0-5 mm), and positive (0 mm). Disease recurrence, disease-free survival and overall survival were evaluated according to the individual resection margins. Disease recurrence occurred in 30.6% of patients with negative, 40.0% with close, and 63.6% with positive resection margins. Significantly shorter disease-free survival and shorter overall survival in patients with positive resection margins was proven. The five-year survival rate was 63.9% in patients with negative, 57.5% with close, and only 13.6% with positive resection margins. The risk of death was 3.27times higher in patients with positive resection margins compared to patients with negative resection margins. Positive resection margins represent anegative prognostic factor, which was also confirmed in our study. There is no unequivocal consensus on the definition of close and negative resection margins and also on the prognostic significance of close resection margins. Factors that may contribute to inaccuracy in the evaluation of resection margins include tissue shrinkage after excision and after fixation of specimens before the histopathological examination. Positive resection margins were associated with asignificantly higher incidence of disease recurrence, shorter disease-free survival and shorter overall survival. When comparing the incidence of recurrence, disease-free survival, and overall survival between patients with close and negative resection margins, the differences were not statistically significant.

One for All, All for One: A Close Look at In-Resin Fluorescence Protocols for CLEM.

Sample preparation is the novel bottleneck for high throughput correlative light and electron microscopy (CLEM). Protocols suitable for both imaging methods must therefore balance the requirements of each technique. For fluorescence light microscopy, a structure of interest can be targeted using: 1) staining, which is often structure or tissue specific rather than protein specific, 2) dye-coupled proteins or antibodies, or 3) genetically encoded fluorescent proteins. Each of these three methods has its own advantages. For ultrastructural investigation by electron microscopy (EM) resin embedding remains a significant sample preparation approach, as it stabilizes the sample such that it withstands the vacuum conditions of the EM, and enables long-term storage. Traditionally, samples are treated with heavy metal salts prior to resin embedding, in order to increase imaging contrast for EM. This is particularly important for volume EM (vEM) techniques. Yet, commonly used contrasting agents (e.g., osmium tetroxide, uranyl acetate) tend to impair fluorescence. The discovery that fluorescence can be preserved in resin-embedded specimens after mild heavy metal staining was a game changer for CLEM. These so-called in-resin fluorescence protocols present a significant leap forward for CLEM approaches towards high precision localization of a fluorescent signal in (volume) EM data. Integrated microscopy approaches, combining LM and EM detection into a single instrument certainly require such an “all in one” sample preparation. Preserving, or adding, dedicated fluorescence prior to resin embedding requires a compromise, which often comes at the expense of EM imaging contrast and membrane visibility. Especially vEM can be strongly hampered by a lack of heavy metal contrasting. This review critically reflects upon the fundamental aspects of resin embedding with regard to 1) specimen fixation and the physics and chemistry underlying the preservation of protein structure with respect to fluorescence and antigenicity, 2) optimization of EM contrast for transmission or scanning EM, and 3) the choice of embedding resin. On this basis, various existing workflows employing in-resin fluorescence are described, highlighting their common features, discussing advantages and disadvantages of the respective approach, and finally concluding with promising future developments for in-resin CLEM.

Taxonomic Identification of Two Poorly Known Lantern Shark Species Based on Mitochondrial DNA From Wet-Collection Paratypes

Etmopteridae (lantern sharks) is the most species-rich family of sharks, comprising more than 50 species. Many species are described from few individuals, and re-collection of specimens is often hindered by the remoteness of their sampling sites. For taxonomic studies, comparative morphological analysis of type specimens housed in natural history collections has been the main source of evidence. In contrast, DNA sequence information has rarely been used. Most lantern shark collection specimens, including the types, were formalin fixed before long-term storage in ethanol solutions. The DNA damage caused by both fixation and preservation of specimens has excluded these specimens from DNA sequence-based phylogenetic analyses so far. However, recent advances in the field of ancient DNA have allowed recovery of wet-collection specimen DNA sequence data. Here we analyse archival mitochondrial DNA sequences, obtained using ancient DNA approaches, of two wet-collection lantern shark paratype specimens, namely Etmopterus litvinovi and E. pycnolepis, for which the type series represent the only known individuals. Target capture of mitochondrial markers from single-stranded DNA libraries allows for phylogenetic placement of both species. Our results suggest synonymy of E. benchleyi with E. litvinovi but support the species status of E. pycnolepis. This revised taxonomy is helpful for future conservation and management efforts, as our results indicate a larger distribution range of E. litvinovi. This study further demonstrates the importance of wet-collection type specimens as genetic resource for taxonomic research.

3D PRINTING AS A TOOL IN ANATOMY TEACHING

Anatomy is essentially a three-dimensional content and learning its structures, through 3D impressions, for example, is of notable importance. In this context, traditional teaching methods, despite being highly effective, still have some limitations. Therefore, 3D printing has been introduced in the teaching of Anatomy, bringing several advantages, such as accuracy, personalized study and easy handling. Based on these premises, the objective of this work was to carry out an integrative review on the use of 3D printing in the teaching of human anatomy. A study was carried out in science direct, PUBMED, Scielo databases between 2010 and 2021 using the following descriptors 3D printing and teaching of anatomy. It was found that among the benefits of using 3D parts, there are: accuracy, durability, ease of production, good cost-benefit ratio and reduction of security risks linked to the fixation of cadaver and plastinated specimens. It was observed that in some studies most students preferred the use of 3D printing to traditional methods. Other studies have shown the importance of the use of 3D printing as a complementary tool to traditional methods of teaching anatomy. It was found that the use of 3D printing as a teaching tool may reduce the demand for bodies and overcome some of the governmental legal and ethical problems in the cadaver study, further studies should be carried out to assess the long-term impact of using 3D printing.

Classifying Pulmonary and Urinary High-grade Neuroendocrine Carcinoma by CK7 Immunohistochemistry.

High-grade neuroendocrine carcinoma (HGNEC) is subclassified into small cell carcinoma (SmCC) and large cell neuroendocrine carcinoma (LCNEC). Although both are clinically aggressive, the SmCC and LCNEC need to have different treatment strategies, and accurate pathologic diagnosis is challenging. We studied a large retrospective cohort (186 cases) of HGNEC of bladder and lung to investigate the abundance of cytokeratin (CK) 7 expression and staining pattern in SmCC and LCNEC. Overall, the pulmonary and urinary HGNEC exhibited several different CK7 staining patterns, including negative staining (n=28), dot-like staining (n=73), partial membranous staining (n=26), and complete membranous staining (n=60). Overall, 88.9% (44/49) of pulmonary SmCC and 88.0% (44/50) of urinary SmCC showed negative or dot-like patterns for CK7, while 90.8% (59/65) of pulmonary LCNEC and 72.7% (16/22) of urinary LCNEC showed partial or complete membranous patterns for CK7 (χ 2 =105.05, P <0.0001). The distinct staining patterns were also present in those mixed SmCC and LCNEC. In addition, the specimen types or fixation did not affect CK7 staining patterns. In conclusion, CK7 has a high differential value for SmCC and LCNEC and could help guide personalized treatment for patients.