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Although many of the structures and organelles of vegetative cells are comparable to those of animal tissues, significant differences between the two kingdoms require modifications in histological techniques for both tissue processing steps and histochemical staining techniques. The authors investigated the challenges of working with plant tissues by collecting various flora to represent the four main plant organs: leaf, stem, root, and flower/fruit. Triplicate samples for each specimen were placed into formalin for paraffin embedding, placed into formalin for later frozen sections, and used fresh to undergo immediate frozen sectioning. Frozen sections of plant tissues were more difficult to obtain than formalin-fixed paraffin-embedded (FFPE) sections, exhibited tissue loss during staining, and were inferior morphologically to FFPE sections. Although, historically, plant tissue fixation and processing has employed several different reagents compared with those used in animal tissue processing and took significantly longer times, the current investigation determined reagents and protocols from a modern histology laboratory which processes mammalian tissues can be applied to plant tissue processing with only slight modifications in respect to reagent timing. Additionally, staining techniques were compared and while it is well known that plant cell walls stain well with safranin O, the current investigation determined the uptake of safranin O can be accelerated by incubating at 60°C. [ABSTRACT FROM AUTHOR]

Background Autofluorescence (AF)–Raman microspectroscopy is a technology that can detect residual basal cell carcinoma (BCC) on the resection margin of fresh, surgically excised tissue specimens. The technology does not require tissue fixation, staining, labelling or sectioning, and provides quantitative diagnosis maps of the surgical margins in 30 min. Objectives To determine the accuracy of the AF–Raman instrument in detecting incomplete BCC excisions during Mohs micrographic surgery (MMS), using histology as the reference standard. Methods Skin layers from 130 patients undergoing MMS at the Nottingham University Hospitals NHS Trust (September 2022–July 2023) were investigated with the AF–Raman instrument. The layers were measured when fresh, immediately after excision. The AF–Raman results and the intraoperative assessment by Mohs surgeons were compared with a postoperative consensus-derived reference produced by three dermatopathologists. The sensitivity, specificity, and positive and negative predictive values were calculated. The study was registered with ClinicalTrials.gov (NCT03482622). Results AF–Raman analysis was successfully completed for 125 of 130 layers and, on average, covered 91% of the specimen surface area, with the lowest surface area covered being 87% for the eyelid and the highest being 94% for forehead specimens. The AF–Raman instrument identified positive margins in 24 of 36 BCC-positive cases [67% sensitivity, 95% confidence interval (CI) 49–82] and negative margins in 65 of 89 BCC-negative cases (73% specificity, 95% CI 63–82). Only one of 12 false-negative cases was caused by misclassification by the AF–Raman algorithm. The other 11 false-negatives cases were a result of no valid Raman signal being recorded at the location of the residual BCC due to either occlusion by blood or poor contact between tissue and the cassette window. The intraoperative diagnosis by Mohs surgeons identified positive margins in 31 of 36 BCC-positive cases (86% sensitivity, 95% CI 70–95) and negative margins in 79 of 89 BCC-negative cases (89% specificity, 95% CI 81–95). Conclusions The AF–Raman instrument has the potential to provide intraoperative microscopic assessment of surgical margins in BCC surgery. Further improvements are required for tissue processing, to ensure complete coverage of the surgical specimens. [ABSTRACT FROM AUTHOR]

• Multiparameter DiOlistics facilitates contextual assessment of neuronal networks. • Tissue fixation enables higher throughput multiparameter staining of tissues. • Fixed tissue DiOlistics provides reliable imaging of dendrites and dendritic spines. DiOlistic labelling is a robust, unbiased ballistic method that utilises lipophilic dyes to morphologically label neurons. While its efficacy on freshly dissected tissue specimens is well-documented, applying DiOlistic labelling to stored, fixed brain tissue and its use in polychromatic multi-marker studies poses significant technical challenges. Here, we present an improved, step-by-step protocol for DiOlistic labelling of dendrites and dendritic spines in fixed mouse tissue. Our protocol encompasses the five key stages: Tissue Preparation, Dye Bullet Preparation, DiOlistic Labelling, Confocal Imaging, and Image Analysis. This method ensures reliable and consistent labelling of dendritic spines in fixed mouse tissue, combined with increased throughput of samples and multi-parameter staining and visualisation of tissue, thereby offering a valuable approach for neuroscientific research. [ABSTRACT FROM AUTHOR]

Objective: This study examined the long-term outcomes of scleral-sutured fixated intraocular lenses (SSIOLs) in patients with uveitis. Patients and Methods: Retrospective, consecutive review of uveitis patients with SSIOL fixation from January 2017 to December 2020. SSIOL techniques included four-point fixation with the Akreos A060 or enVista MX60 (Bausch + Lomb) lens, or Hoffman's pockets rescue. Results: Thirteen eyes of 13 patients received an SSIOL (9 AO60, 2 MX60, 2 Hoffman's pockets). Diagnoses included pan- (9), anterior (2), and posterior uveitis (2). Average LogMAR best-corrected visual acuity pre- and postoperatively was 1.01 (Snellen ∼20/200) and 0.50 (Snellen ∼20/60), respectively (P = 0.003). No patients had postoperative SSIOL dislocation or conjunctival suture erosion. Six patients (46%) had uveitis flares postoperatively. Average follow-up was 50.2 months (range = 36.8 to 67.5). Conclusion: This series demonstrates a 0% dislocation and suture exposure rate. Risks of uveitis flares postoperatively are high despite aggressive perioperative control but are manageable with current treatments; therefore, patients must be continually monitored. [Ophthalmic Surg Lasers Imaging Retina 2024;55:443–447.] [ABSTRACT FROM AUTHOR]

Background: The image quality of tissues obtained with high-resolution imaging systems depends on several factors including the quality of sections prepared by different tissue processing methods. Fresh frozen sections and formalin-fixed paraffin-embedded (FFPE) sections are the two routinely employed methods in histopathological examination having their own merits and demerits. Aims and Objectives: We tested the feasibility of a hybrid tissue processing method that could reduce the processing time without compromising the quality of processed and stained tissues. Materials and Methods: Bovine pancreatic tissue was utilized to standardize the process. The steps included fixation of tissue in formalin, followed by embedding in optimal cutting temperature compound and then cryo-sectioning to obtain 50 pm slices for immunostaining. The prepared thick slices were immunostained with islet markers and assessed by confocal imaging. Results: The images revealed that the staining quality was comparable with FFPE sections, though the tissue processing time was significantly curtailed. The modified Leica microsystems scoring system revealed a nearly 80% overall total score for the processed tissue specimens, indicating acceptable levels of quality of the tissues. We also observed that the staining quality of these formalin-fixed cryo-slices was preserved without tissue deterioration for up to 1 month when stored in formalin, indicating its storage potential for future applications. Conclusion: The proof of concept of the hybrid tissue processing method tested in this study will encourage researchers to extrapolate its use to the human pancreas, other solid organs, and other staining techniques for evaluating its general utility. [ABSTRACT FROM AUTHOR]

The growing scale and dimensionality of multiplexed imaging require reproducible and comprehensive yet user-friendly computational pipelines. TRACERx-PHLEX performs deep learning-based cell segmentation (deep-imcyto), automated cell-type annotation (TYPEx) and interpretable spatial analysis (Spatial-PHLEX) as three independent but interoperable modules. PHLEX generates single-cell identities, cell densities within tissue compartments, marker positivity calls and spatial metrics such as cellular barrier scores, along with summary graphs and spatial visualisations. PHLEX was developed using imaging mass cytometry (IMC) in the TRACERx study, validated using published Co-detection by indexing (CODEX), IMC and orthogonal data and benchmarked against state-of-the-art approaches. We evaluated its use on different tissue types, tissue fixation conditions, image sizes and antibody panels. As PHLEX is an automated and containerised Nextflow pipeline, manual assessment, programming skills or pathology expertise are not essential. PHLEX offers an end-to-end solution in a growing field of highly multiplexed data and provides clinically relevant insights. Imaging dozens of proteins in situ and at large scales increases the complexity of data analysis. Here, the authors develop an end-to-end solution for multiplexed image analysis that facilitates data interpretation for clinically relevant insights. [ABSTRACT FROM AUTHOR]

Background: The way of testicular tissue fixation directly affects the correlation and structural integrity between connective tissue and seminiferous tubules, which is essential for the study of male reproductive development. This study aimed to find the optimal fixative and fixation time to produce high-quality testicular histopathological sections, and provided a suitable foundation for in-depth study of male reproductive development with digital pathology technology. Methods: Testes were removed from both sides of 25 male C57BL/6 mice. Samples were fixed in three different fixatives, 10% neutral buffered formalin (10% NBF), modified Davidson's fluid (mDF), and Bouin's Fluid (BF), for 8, 12, and 24 h, respectively. Hematoxylin and eosin (H&E) staining, periodic acid Schiff-hematoxylin (PAS-h) staining, and immunohistochemistry (IHC) were used to evaluate the testicle morphology, staging of mouse seminiferous tubules, and protein preservation. Aperio ScanScope CS2 panoramic scanning was used to perform quantitative analyses. Results: H&E staining showed 10% NBF resulted in an approximately 15–17% reduction in the thickness of seminiferous epithelium. BF and mDF provided excellent results when staining acrosomes with PAS-h. IHC staining of synaptonemal complexes 3 (Sycp3) was superior in mDF compared to BF-fixed samples. Fixation in mDF and BF improved testis tissue morphology compared to 10% NBF. Conclusions: Quantitative analysis showed that BF exhibited a very low IHC staining efficiency and revealed that mouse testes fixed for 12 h with mDF, exhibited morphological details, excellent efficiency of PAS-h staining for seminiferous tubule staging, and IHC results. In addition, the morphological damage of testis was prolonged with the duration of fixation time. [ABSTRACT FROM AUTHOR]

Corrective osteotomy allows to improve joint loading, pain and function. In complex deformities, the biggest challenge is to define the optimal surgical solution, while considering anatomical, technical and biomechanical factors. While the single-cut osteotomy (SCOT) and focal dome osteotomy (FDO) are well-established treatment options, their mathematical relationship remain largely unclear. The aim of the study was (1) to describe the close mathematical relationship between the SCOT and FDO and (2) to analyze and introduce a novel technique—the stepped FDO—as a modification of the classic FDO. The mathematical background and relationship of SCOT and FDO are described for the example of a femoral deformity correction and visualized using a 3D surface model taking into account the benefits for the clinical application. The novel modifications of the stepped FDO are introduced and its technical and clinical feasibility demonstrated. Both, SCOT and FDO, rely on the same deformity axis that defines the rotation axis k for a 3D deformity correction. To achieve the desired correction using a SCOT, the resulting cutting plane is perpendicular to k, while using a FDO will result in a cylindrical cut with a central axis parallel to k. The SCOT and FDO demonstrate a strong mathematical relation, as both methods rely on the same deformity axis, however, resulting in different cutting planes. These characteristics enable a complementary use when defining the optimal type of osteotomy. This understanding enables a more versatile planning approach when considering factors as the surgical approach, biomechanical characteristics of fixation or soft tissue conditions. The newly introduced stepped FDO facilitates an exact reduction of the bone fragments and potentially expands the clinical applicability of the FDO. [ABSTRACT FROM AUTHOR]

Objective: The treatment of talar neck and/or body fractures is known to be difficult and challenging, with significant impact on the long‐term functional outcome for the patient. The optimal management, including the choice of surgical approaches and implants, are still under constant discussion. The purpose of the study was to investigate the clinical effects of lateral mini‐plate combined with medial lag screws for the treatment of complicated central talar fractures. Methods: The data of eight patients with complex central talus fractures treated between June 2019 and January 2021 were retrospectively analyzed. There were six males and two females, ranging in age from 15 to 66 years, with an average age of 37.4 years. There were three cases on the left and five cases on the right. All fractures were comminuted, including talar neck with talar body fracture in seven cases and talar body comminuted with subluxation of subtalar joint in one case. All patients were treated with the anteromedial combined anterolateral approach, lateral talar mini‐plate fixation and medial lag screw fixation. Fracture reduction quality, union time, and complications were recorded, and functional outcomes were evaluated using the American Orthopedic Foot & Ankle Society (AOFAS) scoring system. Results: The time from injury to surgery was 1–6 days, with an average of 3.38 days. The follow‐up period was 34–53 months (mean 44.88 months). All fractures healed with a mean healing time of 16.75 weeks (13–23 weeks). Anatomical reduction was observed in six cases and near in two cases. After operation, there was no loosening or breakage of implant, loss of fracture reduction, and irritation of skin and soft tissue by internal fixation. The average AOFAS score was 87.38 (48–100), with excellent five cases, good two cases and poor one case, and the excellent and good rate was 87.5%. Superficial skin necrosis in one surgical incision healed after dressing exchange. No deep infection occurred. One case (1/8, 12.5%) developed avascular necrosis of the talus without collapse. Posttraumatic arthritis was found in four cases (4/8, 50%). Conclusion: The utilization of lateral mini‐plates in combination with medial screws for treating complex central talar fractures results in satisfactory reduction and stable fixation, mitigating complications associated with poor reduction. However, due to the absence of an anatomical mini‐plate, pre‐contouring is necessary when applying the lateral plate. This demands a surgeon's thorough familiarity with the anatomical morphology of the talus and proficiency in surgical techniques. Posttraumatic arthritis is the most common complication of complex central talar fractures. [ABSTRACT FROM AUTHOR]

Introduction: Tissue fixation is an essential step in the microscopic preparation of tissues to preserve them by preventing autolysis, bacterial putrefaction, and increasing the tissue's refractive index. The conventional fixative used is 40% formaldehyde. Short-term exposure to formaldehyde can cause irritation to the eyes and respiratory tract, leading to breathlessness and coughing. The International Agency for Research on Cancer (IARC) has classified formaldehyde as a Class 1 human carcinogen capable of potentially causing various neoplasms, including nasopharyngeal carcinoma. Therefore, an innovative approach is being explored to replace formalin with harmless and eco-friendly jaggery. Jaggery possesses cytoprotective, antioxidant, and tissue-preservative properties. At a low pH, the fructose in jaggery breaks down into aldehydes, which cross-link with tissue amino acids, resulting in tissue fixation similar to formaldehyde. Aim: To compare the histological and gross morphological features of tissues fixed in formalin and jaggery. Materials and Methods: This cross-sectional study was conducted in Department of Pathology of a tertiary care centre. Surgical specimens obtained fresh, which were not suspicious of malignancy, were included. A sample size of 23 was selected. Surgical specimens already placed in formalin were excluded. A 30% jaggery solution was prepared by dissolving 300 grams of finely powdered jaggery in 1000 mL of distilled water, which was then filtered using filter paper. A 40% formaldehyde solution was used to prepare a 10% formalin fixative. Tissue bits from each specimen were cut into two halves and placed in formalin and jaggery separately. After 24 hours of fixation, the tissue bits were evaluated for gross morphological features, including tissue shrinkage, consistency, and colour. Tissue shrinkage was classified as mild, moderate, or marked, while consistency ranged from soft to firm to hard. The colour of the specimens varied case by case. Stained slides from jaggeryfixed and formalin-fixed tissues were assessed for histological parameters, such as nuclear details, cytoplasmic details, cellular outline, and overall staining quality. A blinded method was used to compare the stained slides using a microscope. Ratings were assigned to each case on a scale of 1-4. The data were statistically analysed using R software. Results: When comparing the formalin-fixed and jaggery-fixed specimens, no significant differences were observed in gross morphological features. All jaggery-fixed specimens appeared brown grossly. Histological features also showed no significant difference (p-value>0.05) except for cytoplasmic details. Therefore, it was observed that tissue preservation using the jaggery solution was comparable to that of formalin. Conclusion: The tissue-preservative properties of jaggery are on par with formalin. Hence, jaggery solution can be used as an eco-friendly substitute for formalin. Further research with larger sample sizes can pave the way for the effective replacement of hazardous formalin with natural jaggery. [ABSTRACT FROM AUTHOR]

Background: Tissue-based comprehensive genomic profiling (CGP) is increasingly being employed for genotype-directed therapies in patients with advanced cancer. However, tissue availability may limit their potential applications. In Japan, the cost of cancer gene panel tests is covered by public insurance for patients diagnosed with advanced solid tumors once in their lifetime. Therefore, it is essential to improve the success rate (reportability) and accuracy of CGP tests. The purpose of this study was to identify the factors associated with efficient and accurate CGP testing using relevant information obtained from real-world data. Methods: This study included 159 samples analyzed using tumor-only panel FoundationOne® CDx cancer genome profiling (F1CDx) and 85 samples analyzed using matched-pair panel OncoGuide™ NCC Oncopanel system (NCCOP) at St. Marianna University Hospital. Sample characteristics (fixation conditions, storage period, histology, tumor cell ratio, and genomic tumor cell content), CGP performance, and quality control status were evaluated across all 244 tested samples. Results: In 237/244 samples (97.1%), CGP testing results were successfully obtained [F1CDx, 99.4% (158/159) and NCCOP, 92.9% (79/85)]. An increased number of fibroblasts, inflammatory cells, and necrotic tumor cells, long-term storage, and/or prolonged fixation of tissue sections were involved in the unreported results and/or qualified CGP results. In addition, a negative correlation between median insert size values and ΔΔCq was observed in the NCCOP system. Conclusion: We identified various factors associated with efficient and accurate CGP testing using relevant information obtained from real-world data, suggesting that thorough selection and preparation of tissue sections could optimize CGP and maximize useful information. [ABSTRACT FROM AUTHOR]

Terrestrial plants from the very limits of life are likely to harbor genes that confer an advantage in human space exploration. These plants are seemingly capable of performing mission critical functions in spaceflight and on extraterrestrial farms while informing directed gene manipulation in target plant species. However, their adaptations to physiologically extreme habitats may hinder the efficacy of routine laboratory techniques for model plants. We here present the development of Antarctic moss Ceratodon purpureus payload and flight operations for the ANT1 Radiation Tolerance Experiment with Moss in Orbit on the Space Station (ARTEMOSS) experiment to the International Space Station (ISS) given limited physical space and crew time. We demonstrate that the hydrophobic surface of Antarctic moss impedes chemical tissue fixation and precludes the use of RNAlater coupled with payload hardware deployed in standard plant spaceflight experiments. We show that deep-freezing the moss tissue on Petri plates provides adequate tissue fixation and allows the extraction of high-quality RNA suitable for gene expression profiling. We replaced hardware with stacks of Petri plates housing Antarctic moss and chemical fixation with deep-freezing in a cryogenic GLACIER freezer. Our design can be translated to other plant species to expand current experimentation techniques with plants from extreme terrestrial environments in order to advance human space exploration. [ABSTRACT FROM AUTHOR]

Proper fixation and staining of biological samples for electron microscopy (EM) is critical for preserving ultrastructure and providing sufficient contrast for imaging. Depending on experimental goals, different fixation and post-fixation staining methods can be chosen to elucidate the details needed to answer scientific questions. In this guide, we discuss the importance of primary fixation of biological samples and then focus on two major post-fixation staining schemes: the "TOU" and the "OTO" methods, with and without modifications, for highlighting specific structures within the tissue being examined. While both methods can be used for transmission (TEM) and scanning electron microscopy (SEM), we discuss which imaging technique is best suited for each method. [ABSTRACT FROM AUTHOR]

CNS tumor diagnosis in dogs often relies on immunohistochemistry (IHC) given similar histologic features among tumors. Most CNS tissue samples encountered by diagnostic pathologists are collected during autopsy, and postmortem specimens can be susceptible to autolysis and prolonged formalin fixation, both of which have the potential to influence IHC results and interpretation. Here we evaluated the effects of experimentally controlled autolysis induced by delayed tissue fixation (sections of brain held for 2, 4, 8, 12, 24, 48, and 72 h in 0.9% NaCl at either room temperature or 37°C prior to fixation) as well as the effects of prolonged formalin fixation times (1 wk, 1 mo, 2 mo) on a panel of 8 IHC markers (CNPase, GFAP, Iba1, OLIG2, PGP9.5, MAP2, NeuN, synaptophysin) relevant to brain tumor diagnosis. Prolonged fixation of up to 2 mo had no detrimental effect on any immunomarker except NeuN, which had reduced immunolabeling intensity. Delayed fixation led to autolytic changes as expected, on a gradient of severity corresponding to increased time in saline prior to fixation. Several immunomarkers should be used with caution (CNPase, OLIG2) or avoided entirely (MAP2, NeuN) in markedly autolyzed brain and brain tumor tissues. Our results suggest that autolysis has minimal effect on most immunomarkers, but that advanced autolysis may cause a loss of specificity for GFAP, MAP2, and PGP9.5, a loss of intensity of CNPase and OLIG2, and loss of labeling with MAP2 and NeuN. Prolonged fixation affected only NeuN, with mildly decreased intensity. [ABSTRACT FROM AUTHOR]

Background: Tissue fixation is a crucial step to preserve the tissues in a life-like state with minimal disruption to its cellular and chemical composition for histopathological examination. The search for an effective alternate tissue fixative to the routinely used formaldehyde has gained interest as constant exposure to formaldehyde has proven to be toxic. Honey, an organic substance with high acidity and hygroscopic nature, exhibits tissue fixative properties and has been used in the present study. The present study aimed to standardize honey as a tissue fixative for histopathology by comparing it with formalin. Materials and Methods: In vitro study Oral tissue samples of goat were fixed in 10% honey and 10% formalin solution, respectively, for 24--48 h, followed by routine tissue processing and microscopic examination of 37 slides per group. 2200 epithelial cells (1100 per group) were selected for the computer-aided morphometric image analysis (Fiji-Image J) by three observers. Cell area (CA), cell perimeter (CP), nuclear area (NA), nuclear perimeter (NP), cytoplasmic area (Cyt A), and nuclear-cytoplasmic ratio were the parameters studied. Mann--Whitney U-test (STATA/IC version 16) for inter-group comparison was done and P < 0.05 was considered statistically significant. Results: The probability of epithelial cells in the honey-fixed group to have greater NA, NP, and N/C ratio was about 50%--60%. The probability of epithelial cells in formalin-fixed tissues to have greater CA, CP, and Cyt A was about 70%. Conclusion: Honey is a better nuclear fixative than formalin. Cytoplasmic shrinkage of epithelial cells should be taken into consideration while fixing tissues with honey. [ABSTRACT FROM AUTHOR]

Traditional histochemical staining of post-mortem samples often confronts inferior staining quality due to autolysis caused by delayed fixation of cadaver tissue, and such chemical staining procedures covering large tissue areas demand substantial labor, cost and time. Here, we demonstrate virtual staining of autopsy tissue using a trained neural network to rapidly transform autofluorescence images of label-free autopsy tissue sections into brightfield equivalent images, matching hematoxylin and eosin (H&E) stained versions of the same samples. The trained model can effectively accentuate nuclear, cytoplasmic and extracellular features in new autopsy tissue samples that experienced severe autolysis, such as COVID-19 samples never seen before, where the traditional histochemical staining fails to provide consistent staining quality. This virtual autopsy staining technique provides a rapid and resource-efficient solution to generate artifact-free H&E stains despite severe autolysis and cell death, also reducing labor, cost and infrastructure requirements associated with the standard histochemical staining. Conventional staining of post-mortem samples can be affected by several factors, including tissue autolysis. Here, the authors demonstrate a virtual staining tool using a trained neural network to turn autofluorescence images of label-free autopsy tissue into brightfield equivalent images. [ABSTRACT FROM AUTHOR]

• Evidence-based analysis of peri‑implant gas accumulation (PIGA). • Postoperative PIGA: common for musculoskeletal Mg implants. • Clinical relevance: potentially associated surgical complications. • Radiological techniques: for quantitative and longitudinal analysis. • Standardized evaluation needed for developing Mg implants. Historically, the rapid degradation and massive gas release from magnesium (Mg) implants resulted in severe emphysema and mechanical failure. With the advent of new alloys and surface treatment methods, optimized Mg implants have re-entered clinics since last decade with reliable performance. However, the optimization aims at slowing down the degradation process, rather than exemption of the gas release. This study involved a systematic evaluation of current preclinical and clinical evidence, regarding the physical signs, symptoms, radiological features, pathological findings and complications potentially associated with peri‑implant gas accumulation (PIGA) after musculoskeletal Mg implantation. The literature search identified 196 potentially relevant publications, and 51 papers were enrolled for further analysis, including 22 preclinical tests and 29 clinical studies published from 2005 to 2023. Various Mg-based materials have been evaluated in animal research, and the application of pure Mg and Mg alloys have been reported in clinical follow-ups involving multiple anatomical sites and musculoskeletal disorders. Soft tissue and intraosseous PIGA are common in both animal tests and clinical follow-ups, and potentially associated with certain adverse events. Radiological examinations especially micro-CT and clinical CT scans provide valuable information for quantitative and longitudinal analysis. While according to simulation tests involving Mg implantation and chemical processing, tissue fixation could lead to an increase in the volume of gas cavity, thus the results obtained from ex vivo imaging or histopathological evaluations should be interpreted with caution. There still lacks standardized procedures or consensus for both preclinical and clinical evaluation of PIGA. However, by providing focused insights into the topic, this evidence-based study will facilitate future animal tests and clinical evaluations, and support developing biocompatible Mg implants for the treatment of musculoskeletal disorders. [ABSTRACT FROM AUTHOR]

Introduction: The tibia, the second largest bone in the human body, is largely enveloped by subcutaneous fat and skin, rendering it vulnerable to fractures and soft tissue injuries. This unique anatomical configuration contributes to a heightened susceptibility to open fractures. Notably, an aggressive treatment protocol involving radical debridement, internal fixation, and immediate or early soft tissue coverage with muscle flaps has demonstrated improved rates of bony union and reduced incidences of infection. Aim: To assess the clinical outcomes of early bony fixation and soft tissue coverage in Type IIIB open proximal tibia fractures. Materials and Methods: This cross-sectional observational study was conducted at the Department of Orthopaedics, BRD Medical College, in Gorakhpur, Uttar Pradesh, India. Total of 30 cases were enrolled between August 2021 and November 2022. The parameters assessed in the study encompassed patient demographics, including age and gender distribution, the aetiology of fractures, soft tissue coverage outcomes, time to bone union, knee joint movement postsurgery, and the incidence of complications such as superficial infections and chronic osteomyelitis. The analysis was conducted using Statistical Package for Social Sciences (SPSS) version 26.0, primarily employing descriptive statistics to present the results, with percentages and frequencies serving as the primary modes of representation. Results: All cases of Gustilo-Anderson Type IIIB high-grade open tibial fractures were included in this study, with the majority of patients being males 26 (87%) aged between 20 to 40 years. Road traffic accidents accounted for 25 (83.3%), while falls from height were responsible in 5 (16.7%) cases. Assessment of soft tissue coverage revealed 73.33% with good healing, 20% with fair healing, and 6.67% with poor healing. The time to bone union varied, with 10% of cases showing non union. Postsurgery, most cases demonstrated knee joint movement of over 120° (86.67%) and a few cases with movement ranging between 90-120° (13.33%). The incidence of complications was relatively low, with 3 (10%) cases presenting superficial infections and 2 (6.7%) suffering from chronic osteomyelitis. Conclusion: Early bony fixation and soft tissue coverage utilising the medial gastrocnemius flap, along with Split-Thickness Skin Grafting (STSG), demonstrate efficacy in treating high-grade open proximal tibia fractures (Gustilo-Anderson Type IIIB). This approach facilitates expedited bony union and contributes to a reduction in postoperative complications. [ABSTRACT FROM AUTHOR]

Beekeeping plays a crucial role in biodiversity, pollination, commercial farming, and the worldwide agricultural economy. Histopathology, which is an important tool for the investigation of diseases in vertebrates, is not commonly used in honey bees (Apis mellifera). However, histopathology could potentially help the diagnostic investigation of high mortality in bees. We developed a tissue fixation and processing method enabling systematic production of histologic slides adequate for diagnostic and research purposes. Our method uses inexpensive, accessible products and can be realized with conventional pathology laboratory equipment. The quality of histologic slides obtained is similar to those of vertebrate animals processed routinely in pathology laboratories. Histopathology as a diagnostic and research tool will improve the services currently offered to apiarists and could help decrease the mean mortality rate, increase apiarists' profits, and ensure long-term pollination services. [ABSTRACT FROM AUTHOR]

Brain maturation and neurological diseases are intricately linked to microstructural changes that inherently affect the brain's mechanical behavior. Animal models are frequently used to explore relative brain stiffness changes as a function of underlying microstructure. Here, we are using the cuprizone mouse model to study indentation-derived stiffness changes resulting from acute and chronic demyelination during a 15-week observation period. We focus on the corpus callosum, cingulum, and cortex which undergo different degrees of de- and remyelination and, therefore, result in region-specific stiffness changes. Mean stiffness of the corpus callosum starts at 1.1 ± 0.3 kPa in untreated mice, then cuprizone treatment causes stiffness to drop to 0.6 ± 0.1 kPa by week 3, temporarily increase to 0.9 ± 0.3 kPa by week 6, and ultimately stabilize around 0.7 ± 0.1 kPa by week 9 for the rest of the observation period. The cingulum starts at 3.2 ± 0.9 kPa, then drops to 1.6 ± 0.4 kPa by week 3, and then gradually stabilizes around 1.4 ± 0.3 kPa by week 9. Cortical stiffness exhibits less stiffness variations overall; it starts at 4.2 ± 1.3 kPa, drops to 2.4 ± 0.6 kPa by week 3, and stabilizes around 2.7 ± 0.9 kPa by week 6. We also assess the impact of tissue fixation on indentation-based mechanical tissue characterization. On the one hand, fixation drastically increases untreated mean tissue stiffness by a factor of 3.3 for the corpus callosum, 2.9 for the cingulum, and 3.6 for the cortex; on the other hand, fixation influences interregional stiffness ratios during demyelination, thus suggesting that fixation affects individual brain tissues differently. Lastly, we determine the spatial correlation between stiffness measurements and myelin density and observe a region-specific proportionality between myelin content and tissue stiffness. Despite extensive work, the relationship between microstructure and mechanical behavior in the brain remains mostly unknown. Additionally, the existing variation of measurement results reported in literature requires in depth investigation of the impact of individual cell and protein populations on tissue stiffness and interregional stiffness ratios. Here, we used microindentation measurements to show that brain stiffness changes with myelin density in the cuprizone-based demyelination mouse model. Moreover, we explored the impact of tissue fixation prior to mechanical characterization because of conflicting results reported in literature. We observe that fixation has a distinctly different impact on our three regions of interest, thus causing region-specific tissue stiffening and, more importantly, changing interregional stiffness ratios. [Display omitted] [ABSTRACT FROM AUTHOR]

Progress in neuroscience research hinges on technical advances in visualizing living brain tissue with high fidelity and facility. Current neuroanatomical imaging approaches either require tissue fixation (electron microscopy), do not have cellular resolution (magnetic resonance imaging) or only give a fragmented view (fluorescence microscopy). Here, we show how regular light microscopy together with fluorescence labeling of the interstitial fluid in the extracellular space provide comprehensive optical access in real-time to the anatomical complexity and dynamics of living brain tissue at submicron scale. Using several common fluorescence microscopy modalities (confocal, light-sheet and 2-photon microscopy) in mouse organotypic and acute brain slices and the intact mouse brain in vivo, we demonstrate the value of this straightforward 'shadow imaging' approach by revealing neurons, microglia, tumor cells and blood capillaries together with their complete anatomical tissue contexts. In addition, we provide quantifications of perivascular spaces and the volume fraction of the extracellular space of brain tissue in vivo. Brain morphology is complex, heterogenous and miniaturized—and notoriously difficult to visualize. Dembitskaya et al. show how fluorescence 'shadow imaging' gives detailed and comprehensive access to the cellular architecture of the mouse brain in vivo. [ABSTRACT FROM AUTHOR]

Purpose: Reliable and rapid identification of tumor in the margins of breast specimens during breast-conserving surgery to reduce repeat surgery rates is an active area of investigation. Dual-stain difference imaging (DDSI) is one of many approaches under evaluation for this application. This technique aims to topically apply fluorescent stain pairs (one targeted to a receptor-of-interest and the other a spectrally distinct isotype), image both stains, and compute a normalized difference image between the two channels. Prior evaluation and optimization in a variety of preclinical models produced encouraging diagnostic performance. Herein, we report on a pilot clinical study which evaluated HER2-targeted DDSI on 11 human breast specimens. Procedures: Gross sections from 11 freshly excised mastectomy specimens were processed using a HER2-receptor-targeted DDSI protocol shortly after resection. After staining with the dual-probe protocol, specimens were imaged on a fluorescence scanner, followed by tissue fixation for hematoxylin and eosin and anti-HER2 immunohistochemical staining. Receiver operator characteristic curves and area under the curve (AUC) analysis were used to assess diagnostic performance of the resulting images. Performance values were also compared to expression level determined from IHC staining. Results: Eight of the 11 specimens presented with distinguishable invasive ductal carcinoma and/or were not affected by an imaging artifact. In these specimens, the DDSI technique provided an AUC = 0.90 ± 0.07 for tumor-to-adipose tissue and 0.81 ± 0.15 for tumor-to-glandular tissue, which was significantly higher than AUC values recovered from images of the targeted probe alone. DDSI values and diagnostic performance did not correlate with HER2 expression level, and tumors with low HER2 expression often produced high AUC, suggesting that even the low expression levels were enough to help distinguish tumor. Conclusions: The results from this preliminary study of rapid receptor-specific staining in human specimens were consistent with prior preclinical results and demonstrated promising diagnostic potential. [ABSTRACT FROM AUTHOR]

Fluorogenic substrates are emerging tools that enable studying enzymatic processes within their native cellular environments. However, fluorogenic substrates that function within live cells are generally incompatible with cellular fixation, preventing their tandem application with fundamental cell biology methods such as immunocytochemistry. Here we report a simple approach to enable the chemical fixation of a dark‐to‐light substrate, LysoFix‐GBA, which enables quantification of glucocerebrosidase (GCase) activity in both live and fixed cells. LysoFix‐GBA enables measuring responses to both chemical and genetic perturbations to lysosomal GCase activity. Further, LysoFix‐GBA permits simple multiplexed co‐localization studies of GCase activity with subcellular protein markers. This tool will aid studying the role of GCase activity in Parkinson's Disease, creating new therapeutic approaches targeting the GCase pathway. This approach also lays the foundation for an approach to create fixable substrates for other lysosomal enzymes. [ABSTRACT FROM AUTHOR]

Gelatin zymography is widely used to detect gelatinase activity, which is performed on unfixed tissue because it is assumed that fixation inactivates enzymes. However, using fixed tissues has several advantages over using fresh tissues for such prevention of tissue decay, thereby preserving the proteins as well as the morphology and structure of the specimens. In this study, we investigated the effects of the four commonly used fixatives (ethanol, acetone, zinc-based fixative (ZBF), and paraformaldehyde (PFA)) on the gelatinolytic activity in mouse brain tissue. Multiple protocols were employed to extract proteins from the fixed brain tissue. Western blotting and in-gel zymography (IGZ) were used to detect the gelatinase proteins and gelatinolytic activity of the extractions, respectively. In situ zymography (ISZ) revealed that ethanol, acetone, ZBF, and short-time PFA fixation did not inhibit gelatinolytic activity. Neither 1% Triton + 1 M NaCl nor 10% DMSO + 1 M NaCl was effective in extracting proteins from ethanol-, acetone-, ZBF-, or PFA-fixed brain tissues. However, 8 M urea + 4% CHAPS effectively extracted gelatinase proteins from ethanol- and acetone-fixed tissues while retaining the gelatinolytic activity. 2% SDS effectively extracted gelatinase proteins from ethanol-, acetone-, and ZBF-fixed tissues while retaining the gelatinolytic activity. Although 2% SDS + heating extracted gelatinase proteins from ethanol-, acetone-, ZBF-, and even long-term PFA-fixed tissues, the gelatinolytic activity was not retained. Our findings suggest that both ISZ and IGZ can be performed on fixed brain tissue, which is anticipated to be an improvement over the conventionally used gelatin zymography methods. (J Histochem Cytochem 71: 481–493, 2023) [ABSTRACT FROM AUTHOR]

Background: Though multicolour labelling methods allow the routine detection of a wide range of fluorescent (immuno)probe types in molecular cytogenetics, combined applications for the simultaneous in situ detection of proteins and nucleic acids are still sporadic in plant cell biology. A major bottleneck has been the availability of high-quality plant nuclei with a balance between preservation of 3D ultrastructure and maintaining immunoreactivity. The aim of this study was to develop a quick and reliable procedure to prepare plant nuclei suitable for various combinations of immunolabelling and fluorescence in situ hybridisation methods (immunoFISH-GISH). Results: The mechanical removal of the cell wall and cytoplasm, instead of enzymatic degradation, resulted in a gentle, yet effective, cell permeabilisation. Rather than manually releasing the nuclei from the fixed tissues, the procedure involves in-solution cell handling throughout the fixation and the preparation steps as ended with pipetting the pure nuclei suspension onto microscope slides. The optimisation of several critical steps is described in detail. Finally, the procedure is shown to be compatible with immunolabelling, FISH and GISH as well as their simultaneous combinations. Conclusion: A simple plant cell nuclei preparation procedure was developed for combined immunolabelling-in situ hybridisation methods. The main and critical elements of the procedure are: a short period of fixation, incorporation of detergents to facilitate the fixation of tissues and the penetration of probes, tissue grinding to eliminate unwanted cell components, and an optimal buffer to handle nuclei. The procedure is time efficient and is easily transferable without prior expertise. [ABSTRACT FROM AUTHOR]

The aim of the present study was to report the in vivo thickness of the cotyloid fossa at the acetabular ligamentum teres (LT) attachment and investigate the clearance of the obturator neurovascular bundle. Fifty-five consecutive patients undergoing a total hip arthroplasty for hip osteoarthritis were included. The thickness of the cotyloid fossa was measured at the acetabular LT attachment using a standard depth gauge. The minimal distance (clearance) of the obturator neurovascular bundle to the center of the acetabular LT attachment was measured in 7 patients (14 hips) who also underwent a computed tomography angiography. The average thickness of the cotyloid fossa at the acetabular LT attachment was 4.1 ± 2.3 (range: 1–10) mm. The obturator vein was closest to the acetabular LT attachment, but the clearance was more than the defined safe zone of 15 mm in all cases. Based on the current findings, it can be assumed that bone anchors might not be suitable for fixation of the graft in LT reconstruction (LTR) and an alternative implant such as a cortical button should be considered. Acetabular fixation of the graft with a 12-mm cortical button is relatively safe concerning injury to obturator neurovascular structures. The results of the present study provide a better understanding of the cotyloid fossa anatomy and might be relevant for surgeons who perform arthroscopic LTR. [ABSTRACT FROM AUTHOR]

The proper preservation of human brain tissue is an indispensable requirement for post-mortem investigations. Neuroanatomical teaching, neuropathological examination, neurosurgical training, basic and clinical neuroscientific research are some of the possible downstream applications of brain specimens and, although much apart from one another, proper tissue fixation and preservation is a common denominator to all of them. In this review, the most relevant procedures to fixate brain tissue are described. In situ and immersion fixation approaches have been so far the most widespread ways to deliver the fixatives inside the skull. Although most of them rely on the use of formalin, alternative fixative solutions containing lower amounts of this compound mixed with other preservative agents, have been attempted. The combination of fixation and freezing paved the way for fiber dissection, particularly relevant for the neurosurgical practice and clinical neuroscience. Moreover, special techniques have been developed in neuropathology to tackle extraordinary problems, such as the examination of highly infective specimens, as in the case of the Creutzfeldt-Jakob encephalopathy, or fetal brains. Fixation is a fundamental prerequisite for further staining of brain specimens. Although several staining techniques have been developed for the microscopical investigation of the central nervous system, numerous approaches are also available for staining macroscopic brain specimens. They are mostly relevant for neuroanatomical and neuropathological teaching and can be divided in white and gray matter staining techniques. Altogether, brain fixation and staining techniques are rooted in the origins of neuroscience and continue to arouse interest in both preclinical and clinical neuroscientists also nowadays. [ABSTRACT FROM AUTHOR]

Purpose: To evaluate the efficacy and safety of single-incision mini-sling for stress urinary incontinence based on network Meta-analysis. Materials and Methods: We searched PubMed, Embase, and Cochrane libraries from August 2008 to August 2019. Randomized controlled trials comparing two or more indicators of Miniarc (Single Incision Mini-slings), Ajust (Adjustable Single-Incision Sling), C-NDL (Contasure-Needleless), TFS (Tissue Fixation System), Ophria (Transobturator Vaginal Tap), TVT-O (Transobturator Vaginal Tape), and TOT (Trans-obturatortape) in treating female stress urinary incontinence were collected. Results: Totally, 3,428 patients from 21 studies were included. Ajust had the highest subjective cure rate (Rank = 0.52), while Ophira had the worst (Rank = 0.67). TFS had the highest objective cure rate, and the worst was found in Ophira. TFS required the shortest operating time (Rank = 0.40), while TVT-O required the longest operating time (Rank = 0.47). Miniarc had the least bleeding (Rank = 0.47), while TVT-O had the most bleeding (Rank = 0.37). C-NDL had the shortest postoperative hospital stay (Rank=0.77), while Ajust had the longest postoperative hospital stay (Rank = 0.36). For postoperative complications, TFS performed best in groin pain (Rank = 0.84), urinary retention (Rank = 0.78), and repeat surgery (Rank=0.45). TVT-O performed worst in groin pain (Rank = 0.36), and urinary retention (Rank = 0.58). Miniarc had the highest repeat surgery rate (Rank = 0.35). Ajust had the lowest probability of tap erosion (Rank=0.30), while Ophira had the highest tap erosion level (Rank = 0.45). Miniarc showed the greatest advantage in urinary tract infections (Rank = 0.84) and de novo urgency (Rank = 0.60), while C-NDL had the highest incidence of urethral infections (Rank = 0.51). Ophira performed worst in de novo urgency (Rank = 0.60). C-NDL performed the best in sexual intercourse pain (Rank = 0.79) while Ajust was the worst (Rank = 0.49). Conclusion: In view of comprehensive efficacy and safety, we recommend that TFS or Ajust should be selected first for single-incision sling and the application of Ophria should be minimized. [ABSTRACT FROM AUTHOR]

Postmortem magnetic resonance imaging (MRI) can provide a bridge between histological observations and the in vivo anatomy of the human brain. Approaches aimed at the co-registration of data derived from the two techniques are gaining interest. Optimal integration of the two research fields requires detailed knowledge of the tissue property requirements for individual research techniques, as well as a detailed understanding of the consequences of tissue fixation steps on the imaging quality outcomes for both MRI and histology. Here, we provide an overview of existing studies that bridge between state-of-the-art imaging modalities, and discuss the background knowledge incorporated into the design, execution and interpretation of postmortem studies. A subset of the discussed challenges transfer to animal studies as well. This insight can contribute to furthering our understanding of the normal and diseased human brain, and to facilitate discussions between researchers from the individual disciplines. [ABSTRACT FROM AUTHOR]

Immediate implant placement is considered the treatment of choice for single tooth replacement in the esthetic area. However, this treatment is associated with several critical drawbacks related to the inadequate assessment/management of the soft and hard peri‐implant tissues and their subsequent remodeling, resulting in peri‐implant soft‐tissue defects that can lead to impaired esthetic outcomes in time. We describe in detail how the mucogingival approach to immediate implant placement ensures a standard result regardless of the baseline soft‐hard tissue situation. Fully guided implant placement guarantees an adequate three‐dimensional implant placement, the flap design makes it possible to perform bone augmentation with complete visibility of the area being treated, allows soft tissue augmentation with proper fixation of the connective tissue graft, and the placement of an immediate provisional ensures stabilization of the peri‐implant tissues throughout the healing period. [ABSTRACT FROM AUTHOR]

Purpose: Over the past 40 years, advances in the development of anchors and sutures have contributed to the improvement in surgical outcomes for treatment of shoulder instability. Important choices in surgery when treating instability include the use of knotless versus knotted suture anchors, and bony versus soft tissue reconstruction techniques. Methods: A literature review was conducted to evaluate the history of instability of the shoulder and the results of specific fixation techniques including bony and soft tissue reconstructions as well as knotted and knotless suture anchors. Results: As knotless suture anchors have continued to grow in popularity since their development in 2001, many studies have compared this newer technique to that of the standard knotted suture anchors. In general, these studies have demonstrated no difference in patient-reported outcome measures between the two options. Additionally, the choice of bony versus soft tissue reconstructions is patient specific as it depends on the specific pathology or combination of injuries. Conclusion: In each surgery performed for shoulder instability, it is vitally important that we try to restore normal anatomy. The normal anatomy is best established by knotted mattress sutures. However, loop laxity and tear through by the sutures in the capsule can eliminate this restoration, increasing risk of failure. Knotless anchors may allow better soft tissue fixation of the labrum and capsule to the glenoid, but without complete restoration of normal anatomy. [ABSTRACT FROM AUTHOR]

Background: Postmortem fetal magnetic resonance imaging (MRI) has been on the rise since it was proven to be a good alternative to conventional autopsy. Since the fetal brain is sensitive to postmortem changes, extensive tissue fixation is required for macroscopic and microscopic assessment. Estimation of brain maceration on MRI, before autopsy, may optimize histopathological resources. Objective: The aim of the study is to develop an MRI-based postmortem fetal brain maceration score and to correlate it with brain maceration as assessed by autopsy. Materials and methods: This retrospective single-center study includes 79 fetuses who had postmortem MRI followed by autopsy. Maceration was scored on MRI on a numerical severity scale, based on our brain-specific maceration score and the whole-body score of Montaldo. Additionally, maceration was scored on histopathology with a semiquantitative severity scale. Both the brain-specific and the whole-body maceration imaging scores were correlated with the histopathological maceration score. Intra- and interobserver agreements were tested for the brain-specific maceration score. Results: The proposed brain-specific maceration score correlates well with fetal brain maceration assessed by autopsy (τ = 0.690), compared to a poorer correlation of the whole-body method (τ = 0.452). The intra- and interobserver agreement was excellent (correlation coefficients of 0.943 and 0.864, respectively). Conclusion: We present a brain-specific postmortem MRI maceration score that correlates well with the degree of fetal brain maceration seen at histopathological exam. The score is reliably reproduced by different observers with different experience. [ABSTRACT FROM AUTHOR]

Purpose: Current skin imaging modalities, including optical, electron, and confocal microscopy, mostly require tissue fixations that could damage proteins and biological molecules. Live tissue or cell imaging such as ultrasonography and optical coherent microscope may not adequately measure the dynamic spectroscopical changes. Raman spectroscopy has been adopted for skin imaging in vivo, mostly for skin cancer imaging. However, whether the epidermal and dermal thickening in skin could be measured and distinguished by conventional Ramen spectroscopy or the surface‐enhanced Raman scattering (SERS), a rapid and label‐free method for noninvasive measurement remains unknown. Methods: Human skin sections from patients of atopic dermatitis and keloid, which represent epidermal and dermal thickening, respectively, were measured by conventional Ramen spectroscopy. In mice, skin sections from imiquimod (IMQ)‐ and bleomycin (BLE)‐treated mice, which reflect the epidermal and dermal thickening, respectively, were measured by SERS, that incorporates gold nanoparticles to generate surface plasma and enhance Raman signals. Results: Conventional Ramen spectroscopy failed to consistently show the Raman shift in human samples among the different groups. SERS successfully revealed a prominent peak around 1300 cm−1 in the IMQ‐treated skin; and two significant peaks around 1100 and 1300 cm−1 in BLE‐treated group. Further quantitative analysis showed 1100 cm−1 peak was significantly accentuated in the BLE‐treated skin than that in control skin. SERS identified in vitro a similar 1100 cm−1 peak in solutions of collagen, the major dermal biological molecules. Conclusion: SERS distinguishes the epidermal or dermal thickening in mouse skin with rapid and label‐free measures. A prominent 1100 cm−1 SERS peak in the BLE‐treated skin may result from collagen. SERS might help precision diagnosis in the future. [ABSTRACT FROM AUTHOR]

Phosphorylated biomarkers are crucial for our understanding of drug mechanism of action and dose selection during clinical trials, particularly for drugs that target protein kinases, such as DNA-damage-response (DDR) inhibitors. However, tissue fixation conditions needed to preserve DDR-specific phospho-biomarkers have not been previously investigated. Using xenograft tissues and tightly controlled formalin fixation conditions, we assessed how preanalytical factors affect phosphorylated DDR biomarkers pRAD50(Ser635), ɣH2AX(Ser139), pKAP1(Ser824), and non-phosphorylated biomarkers cMYC and ATM. Cold ischemia times ranged from 15 min to 6 hr, and the fixation duration ranged from 24 hr to 4 weeks. Epitopes pRAD50 and pKAP1 appeared the most labile assessed with staining loss after just 15 min of cold ischemia time, while ATM was more robust showing consistent expression up to 1 hr of cold ischemia. Notably, ɣH2AX expression was lost with formalin fixation over 48 hr. The use of core needle biopsies where possible and novel fixation methods such as the 2-step temperature-controlled formalin approach may improve phosphorylated biomarker preservation; however, practical challenges may affect wider clinical application. The most essential tissue-processing step when downstream analysis includes DDR phosphorylated biomarkers is immediate tissue submersion in formalin, without delay, upon excision from the patient, followed by room temperature fixation for 24 hr. [ABSTRACT FROM AUTHOR]

• Immunohistochemical staining intensity is higher in adequately fixated tumor areas. • Immunohistochemical staining intensity is different within tumors, suggesting heterogeneous immunoreactivity. • Independent of tumor fixation, DNA fragments rarely exceeded a length of 300 base pairs. • The pathologist should be aware of the quality of tumor fixation during the interpretation of immunohistochemistry. The objective is to assess the impact of the quality of tissue fixation in surgical pathology on immunohistochemical (IHC) staining and DNA degradation. Twenty-five non-small cell lung cancer (NSCLC) resection specimens were analyzed. After resection, all tumors were processed according to the protocols in our center. In haematoxylin and eosin (H&E) stained tissue slides, adequately- and inadequately fixed tumor areas were microscopically demarcated, based on basement membrane detachment. In 10 IHC stains ALK (clone 5A4), PD-L (clone 22C3), CAM5.2, CK7, c-Met, KER-MNF116, NapsinA, p40, ROS1, TTF1) the immunoreactivity in H-scores was determined in adequately- and inadequately fixed, and necrotic tumor areas. From the same areas DNA was isolated, and DNA fragmentation in base pairs (bp) was measured. H-scores were significantly higher in H&E adequately fixed tumor areas in IHC stains KER-MNF116 (H-score 256 vs 15, p =0.001) and p40 (H-score 293 vs 248, p =0.028). All other stains showed a trend towards higher immunoreactivity in H&E adequately fixed areas. Independent of H&E adequatelty- or inadequately fixed areas, all IHC stains showed significant different IHC staining intensity within tumors, suggesting heterogeneous immunoreactivity (H-scores: PD-L1 123 vs 6, p = 0.001; CAM5.2 242 vs 101, p =<0.001; CK7 242 vs 128, p =<0.001; c-MET 99 vs 20, p =<0.001; KER-MNF116 281 vs 120, p =<0.001; Napsin A 268 vs 130, p = 0.005; p40 292 vs 166, p = 0.008; TTF1 199 vs 63, p =<0.001). DNA fragments rarely exceeded a length of 300 bp, independent of adequate fixation. However, DNA fragments of 300 and 400 bp had higher concentrations in tumors with short fixation delay (<6 h vs >16 h) and short fixation time (<24 h vs >24 h). Impaired tissue fixation of resected lung tumors results in decreased IHC staining intensity in some parts of the tumor. This may impact the reliability of IHC analysis. [ABSTRACT FROM AUTHOR]

Introduction: The original plastination protocol was described as dehydration and defatting with acetone, low temperature forced impregnation with a silicone polymer, and curing with a silicone cross-linker. This protocol was published, and the technique globally developed in multiple anatomy laboratories. However, some environmental, economic, and governmental regulatory issues affect carrying out this technique as was originally described. Our aim is to present an acetone free protocol as an alternative to carry out plastination. Material and methods: Tissue fixation was carried out through immersion in formaldehyde solution at 4% v/v. Isopropyl alcohol was used for low temperature dehydration. Defatting was done using methylene chloride at room temperature. Low temperature tissue impregnation was carried out with S10-S3 Biodur® silicone in a low-pressure media. Curing is divided into "passive curing" and cross-linking with S6 Biodur®. Results: Dry, durable, non-sticky and flexible plastinated specimens were obtained. Carrying out dehydration and defatting with isopropyl alcohol and methylene chloride, respectively, showed a minimal appearance change in every specimen excepting the ones from the central nervous system. Longer passive curing makes the specimens more flexible but reduces the quality of some impregnated solid-organ specimens. Conclusions: this manuscript presents an acetone free protocol that offers satisfactory plastinated specimens which could be used for research, academical and expositional purposes. This protocol deals with some of the main limitations faced following the original protocol. [ABSTRACT FROM AUTHOR]

Purpose: Treatment of pelvic organ prolapse (POP) often requires the use of synthetic mesh. In case of a novel and standardized bilateral apical fixation, both uterosacral ligaments are replaced by polyvinylidene-fluoride (PVDF) tapes. One of the main problems remains the fixation method, which should be stable, but also simple and quick to use. The current study evaluated biomechanical differences between the cervical tape fixation with sutures (group 1), non-absorbable tacks (group 2) and absorbable tacks (group 3) in an in vitro porcine model. Methods: A total of 28 trials, conducted in three groups, were performed on porcine, fresh cadaver uteri. All trials were performed until mesh, tissue or fixation device failure occurred. Primary endpoints were the biomechanical properties maximum load (N), displacement at failure (mm) and stiffness (N/mm). The failure mode was a secondary endpoint. Results: There was a significant difference between all three groups concerning the maximum load. Group 1 (sutures) supported a maximum load of 64 ± 15 N, group 2 (non-absorbable tacks) yielded 41 ± 10 N and group 3 (absorbable tacks) achieved 15 ± 8 N. The most common failure mode was a mesh failure for group 1 and 2 and a fixation device failure for group 3. Conclusion: The PVDF-tape fixation with sutures supports 1.5 times the load that is supported by non-absorbable tacks and 4.2 times the load that is supported by absorbable tacks. Nevertheless, there was also a stable fixation through tacks. Sutures are the significantly stronger and cheaper fixation device but may prolong the surgical time in contrast to the use of tacks. [ABSTRACT FROM AUTHOR]

Vaginal atresia is seen in genetic disorders such as Mayer–Rokitansky–Küster–Hauser (MRKH) syndrome, which can cause significant sexual dysfunction. Current treatments include surgical reconstruction or mechanical dilation of the vaginal canal. Mechanical dilation requires patients to be highly motivated and compliant while surgical reconstruction has high rates of complications. This study evaluated a novel vaginal expansion sleeve (VES) method as an alternative treatment for vaginal atresia. The proprietary cylindrical VES is a spring-like device consisting of polyethylene terephthalate helicoid trusses capped at each end with a fixed diameter resin cap for fixation within tissues. Following the development of the VES and mechanical characterization of the force–length relationships within the device, we deployed the VES in Sprague Dawley rat vaginas anchored with nonabsorbable sutures. We measured the VES length–tension relationships and post-implant vaginal canal expansion ex vivo. Vaginal histology was examined before and after implantation of the VES devices. Testing of 30 mm sleeves without caps resulted in an expansion force of 11.7 ± 3.4 N and 2.0 ± 0.1 N at 50% and 40%, respectively. The implanted 20 mm VES resulted in 5.36 mm ± 1.18 expansion of the vaginal canal, a 32.5 ± 23.6% increase (p = 0.004, Student t test). Histological evaluation of the VES implanted tissue showed a significant thinning of the vaginal wall when the VES was implanted. The novel VES device resulted in a significant expansion of the vaginal canal ex vivo. The VES device represents a unique alternative to traditional mechanical dilation therapy in the treatment of vaginal atresia and represents a useful platform for the mechanical distension of hollow compartments, which avoids reconstructive surgeries and progressive dilator approaches. [ABSTRACT FROM AUTHOR]

Immunohistochemistry (IHC) is the use of antibodies as probes to determine the localization of proteins in a cell or tissue sample. In microscopy, the technique has been in use since the 1940s by pathologists and research scientists. At first glance it appears to be a straightforward procedure. In practice, however, an IHC protocol has several steps where problems can occur, resulting in either false positive or false negative data. IHC can be used in both transmitted light and fluorescence microscopy, and it can be used in combinations to identify and mark multiple proteins of interest. While many IHC techniques have been published, a certain amount of confusion exists as to how to optimize IHC procedures. This series of three articles will describe the steps involved in IHC, the function of each step, and some variations that occur in specific protocols on differing cell and tissue types. It is hoped that a better understanding of these steps will help to guide users in determining the optimal conditions for carrying through IHC protocols. [ABSTRACT FROM AUTHOR]

Recent advances in regenerative technology have made the regeneration of various organs using pluripotent stem cells possible. However, a simpler screening method for evaluating regenerated organs is required to apply this technology to clinical regenerative medicine in the future. We have developed a simple evaluation method using a mouse tooth germ culture model of organs formed by epithelial–mesenchymal interactions. In this study, we successfully established a simple method that controls tissue development in a temperature-dependent manner using a mouse tooth germ ex vivo culture model. We observed that the development of the cultured tooth germ could be delayed by low-temperature culture and resumed by the subsequent culture at 37 °C. Furthermore, the optimal temperature for the long-term preservation of tooth germ was 25 °C, a subnormothermic temperature that maintains the expression of stem cell markers. We also found that subnormothermic temperature induces the expression of cold shock proteins, such as cold-inducible RNA-binding protein, RNA-binding motif protein 3, and serine and arginine rich splicing factor 5. This study provides a simple screening method to help establish the development of regenerative tissue technology using a tooth organ culture model. Our findings may be potentially useful for making advances in the field of regenerative medicine. [ABSTRACT FROM AUTHOR]

Neutral buffered formalin (NBF) is the most common fixative in clinical applications. However, NBF damages proteins and nucleic acids, limiting the quality of proteomic and nucleic acid-based assays. Prior studies have demonstrated that BE70, a fixative of buffered 70% ethanol, has many benefits over NBF but the degradation of proteins and nucleic acids in archival paraffin blocks remain a challenge. Thus, we evaluated the addition of guanidinium salts to BE70 with the hypothesis that this may protect RNA and protein. Guanidinium salt supplemented BE70 (BE70G)-fixed tissue is comparable with that of BE70 via histology and immunohistochemistry. Western blot analysis also revealed that HSP70, AKT, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression signals in BE70G-fixed tissue were higher than those in BE70-fixed tissue. The quality of nucleic acids extracted from BE70G-fixed, paraffin-embedded tissue was also superior, and BE70G provides improved protein and RNA quality at shorter fixation times than its predecessors. The degradation of proteins, AKT and GAPDH, in archival tissue blocks is also decreased with the addition of guanidinium salt to BE70. In conclusion, BE70G fixative improves the quality of molecular analysis with more rapid fixation of tissue and enhanced long-term storage of paraffin blocks at room temperature for evaluation of protein epitopes: [ABSTRACT FROM AUTHOR]

Femoral de-rotational osteotomies are a safe and effective treatment for symptomatic excessive femoral anteversion or retroversion. The author's preferred technique for performing a de-rotational osteotomy is via a subtrochanteric transverse osteotomy with intramedullary nail fixation. We describe a method for guiding femoral de-rotation correction intra-operatively using a bubble inclinometer. [ABSTRACT FROM AUTHOR]

Aims Green bean (Phaseolus vulgaris L.) is a popular vegetable worldwide. The use of beneficial fungi is a simple and effective way to improve the biological nitrogen fixation (BNF) of this leguminous vegetable. Methods and Results A micro-plot was conducted to investigate the enhancement of BNF using 15N natural abundance technology and agronomic performances of green bean caused by wood-rot fungus Ceriporia lacerata HG2011. The results showed the soil for frequently growing green bean featured abundant native rhizobia, and newly inoculated rhizobia may have to compete with them in nodulation and only highly competitive rhizobia can succeed. The addition of C. lacerata HG2011 to the soil increased the population of ammonia oxidizers, nitrifiers, and phosphorus (P)-mobilizing microbes in rhizosphere, accelerated nitrification and P mobilization, creating a favorable soil environment with high P and low ammonia for BNF. Green bean received C. lacerata HG2011 had higher dehydrogenase activity in roots and higher nodulation rate and large nodules. These phenomena implied abundant supplies of adenosine triphosphate, nicotinamide adenine dinucleotide hydrogen, or nicotinamide adenine dinucleotide phosphate hydrogen for BNF in the roots, a large proportion of N2 fixation tissues, and a greater sink for receiving photosynthates. As a result, C. lacerata HG2011 considerably increased the percentage of N derived from the atmosphere, BNF, and plant nutrient uptake (including N, P, and potassium), leading to 15.58%–28.51% of biomass increasment and 9.82%–17.03% of peapod yield increasment along with quality improvement compared with non-fungal application. Conclusions C. lacerata HG2011 increased the nodulation and BNF of green bean, accelerated the nutrient uptake (NPK) and therefore improved the yield and peapod quality of green bean. Significance and Impact of Study The study demonstrates that C. lacerata HG2011 could be used as a biofertilizer for BNF improvement of legumes. [ABSTRACT FROM AUTHOR]

Background: Formaldehyde, widely utilized in anatomic pathology laboratories, has been classified as a carcinogen, yet its exposure patterns in laboratory practice in Nigeria has been scarcely studied. Aim: The study aimed to evaluate exposure patterns in selected anatomic pathology laboratories in Nigeria. Method: Four laboratories from northern parts of Nigeria were selected for the study. The short-term exposure limits/levels (STEL) in parts per million (ppm) were calculated for 15-minute intervals for the period of surgical cut-up daily for 5-working days. Average values for each laboratory were then documented. Result: The average STEL ranged from as low as 0.002 ppm to as high as 1.6 ppm in the laboratories studied and was influenced by concentration of formaldehyde used in specimen preservation, and to some degree, ambient temperature, and humidity during surgical cut-up. Conclusion: There is a need for anatomic pathology laboratories to constantly monitor the levels of formaldehyde emissions in their workspaces and the need to educate clients of the laboratory on the ideal formaldehyde concentration for tissue fixation and the dangers of concentrated formaldehyde usage. [ABSTRACT FROM AUTHOR]

Objectives: The art of microscopy is only appreciable in well-fixed tissues. We conducted this study to determine the effectiveness of Aloe vera as a tissue fixative and compare its results with the natural fixatives already studied in the literature. Materials and Methods: A pilot study was tried out using commercially available fresh chicken and fish with Aloe vera, and then after getting the promising results similar study protocol was carried out using 10-autopsied human tissue. Four natural fixatives-30% jaggery solution, 20% honey solution, 20% sugar solution, 20% Aloe vera solution and 10% formalin were used for fixation in the study. Fixation of tissues was carried out at room temperature for 24 h. All pre- and postfixation measurements were recorded using stereomicroscope and its software. The difference between pre-and postfixation was calculated and later, all pieces were kept for routine tissue processing followed by routine staining. The tissue sections were assessed for quality, and the whole procedure was blinded among three oral pathologists who scored them. Results: The mean percentage of shrinkage in each bit with different reagents was calculated. The shrinkage seen with 10% formalin and 20% Aloe vera were more likely similar. Among all the natural fixatives, qualitatively also Aloe vera excelled and its results were comparable to that of formalin. Conclusion: The use of Aloe vera in the present study as fixative is the first of its kind, as exhaustive search of the literature shows only its use as transport media in dentistry. [ABSTRACT FROM AUTHOR]

Objectives: Good-quality nucleic acid extraction from formalin-fixed, paraffin-embedded (FFPE) specimens remains a challenge in molecular-oncopathology practice. This study evaluates the efficacy of an in-house developed FFPE extraction buffer compared with other commercially available kits.Methods: Eighty FFPE specimens processed in different surgical pathology laboratories formed the study sample. DNA extraction was performed using three commercial kits and the in-house developed FFPE extraction buffer. DNA yield was quantified by a NanoDrop spectrophotometer and Qubit Fluorometer, and its purity was measured by the 260/280-nm ratio. A fragment analyzer system was used for accurate sizing of DNA fragments of FFPE DNA. The downstream effects of all extraction methods were evaluated by polymerase chain reaction (PCR) and Sanger sequencing.Results: In comparison with the commercial kits, the in-house buffer yielded higher DNA quantity and quality number (P < .0001). In addition, DNA integrity and fragment size were preserved in a significantly greater number of samples isolated with the in-house buffer (P < .05). The target PCR amplification rate with the in-house buffer extracted samples was also significantly higher, with 98% of the samples showing interpretable sequencing results.Conclusions: The in-house developed FFPE extraction buffer performed superior to other methods in terms of suitability for downstream applications, time, cost-efficiency, and ease of performance. [ABSTRACT FROM AUTHOR]

A bio-integrative fiber-reinforced implant (OSSIOfiber® Hammertoe Fixation Implant, OSSIO Ltd., Caesarea, Israel) for proximal interphalangeal joint (PIPJ) correction-arthrodesis showed partial bio-integration at 1-year follow-up (1FU) in a previous study. The study was prolonged to assess the bio-integration at 2-year-follow-up (2FU). Twenty-four patients with proximal interphalangeal joint (PIPJ) correction-arthrodesis using the fiber-reinforced implant and analysed at 1FU, completed 2FU. Follow-up included clinical examination, patient reported outcomes, radiographs, MRI and bio-integration scoring. Results were compared between the 1FU and 2FU (paired t-test). Radiographs confirmed fusion in 96 % (n = 23) at 2FU (1FU, 92 % (n = 22)). Implant was no longer visible in 21 % (n = 5), partially visible in 33 % (n = 8), and fully visible in 46 % (n = 11)(1FU, fully visible 100 % (n = 24)). The border between implant and surrounding bone was scored not visible in 88 % (n = 21) and partially visible in 12 % (n = 3) (1FU, border partially visible 100 % (n = 24)). There were no cyst formation or fluid accumulation findings 1FU/2FU. Mild bone edema was detected in 4 % (n = 1) (1FU, 29 % (n = 7)). None of the edema findings were considered as adverse implant related. The mean bio-integration score was 9.71 ± 0.69 at 2FU (1FU, 7.71 ± 0.46). The parameters of border between implant and bone and bone edema further improved at the 2FU compared to the 1FU, total bio-integration score was also higher at 2FU than 1FU (each p < 0.05). This study demonstrates 96 % PIPJ fusion rate and increased bio-integration from 1FU to 2FU, reaching advanced bio-integration of the fiber-reinforced implant at 2FU. [ABSTRACT FROM AUTHOR]

RNA sequencing (RNA-seq) coupled with laser capture microdissection (LCM) is a powerful tool for transcriptomic analysis in unfixed fresh-frozen tissues. Fixation of ocular tissues for immunohistochemistry commonly involves the use of paraformaldehyde (PFA) followed by embedding in Optimal Cutting Temperature (OCT) medium for long-term cryopreservation. However, the quality of RNA derived from such archival PFA-fixed/OCT-embedded samples is often compromised, limiting its suitability for transcriptomic studies. In this study, we aimed to develop a methodology to extract high-quality RNA from PFA-fixed canine eyes by utilizing LCM to isolate retinal tissue. We demonstrate the efficacy of an optimized LCM and RNA purification protocol for transcriptomic profiling of PFA-fixed retinal specimens. We compared four pairs of canine retinal tissues, where one eye was subjected to PFA-fixation prior to OCT embedding, while the contralateral eye was embedded fresh frozen (FF) in OCT without fixation. Since the RNA obtained from PFA-fixed retinas were contaminated with genomic DNA, we employed two rounds of DNase I treatment to obtain RNA suitable for RNA-seq. Notably, the quality of sequencing reads and gene sets identified from both PFA-fixed and FF tissues were nearly identical. In summary, our study introduces an optimized workflow for transcriptomic profiling from PFA-fixed archival retina. This refined methodology paves the way for improved transcriptomic analysis of preserved ocular tissue, bridging the gap between optimal sample preservation and high-quality RNA data acquisition. [ABSTRACT FROM AUTHOR]

Pelvic organ prolapse (POP) is a condition with an increasing incidence rate, affecting more than 40% of women over 50 years old. There are three levels of pelvic floor support, described by Delancey. The individual impact of each of them leads to the appearance of prolapse in a certain compartment (anterior, posterior or apical), the therapeutic conduct being individualized in each situation. In the case of symptomatic apical POP or after the failure of conservative treatment, the treatment of choice is the surgical one, with hysterectomy in the first place, followed by pelvic reconstruction procedures with the preservation of the uterus. These procedures can be performed through a vaginal approach, if access allows, or transabdominally, classically or through laparoscopy. Additionally, there are numerous procedures for ligamentous suspension of the remaining vaginal apex, thus having the freedom to combine different approaches with different procedures, with the aim of a lasting result. Regarding the recurrence rate, comparing vaginal hysterectomy followed by ligament fixation with native tissue with hysteropreservation interventions, there is a significant decrease in POP recurrence after hysterectomy. However, compared in general, no long-term difference in pelvic organ prolapse recurrence is observed. Moreover, being an invasive procedure that requires extensive dissection of the tissues, there is a longer duration of the operating time, with an above average hemorrhage in case of hysterectomy, the patients having a longer duration of hospitalization. The decision regarding the chosen surgical option must be taken together with the patient, after presenting the advantages and disadvantages of each method, as well as the evaluation of the biological state and the risks of a prolonged surgical intervention. An important factor is the existence of some gynecological background (metrorrhagia, endometrial polyps, uterine fibroids), an aspect that pleads for the excision of the uterus. [ABSTRACT FROM AUTHOR]