Formalin Fixation Research Articles

The clinical significance of the features of the morphological study and immunohistochemical determination of pancytokeratin in the lymph nodes of the central zone in papillary thyroid cancer

Introduction. papillary thyroid cancer is increasingly being detected at early stages when regional and distant metastases are absent per clinical examination. However, lymph nodes of the central zone can carry hidden metastases. frequency of such metastases is 22.3–46.7 %. maximally accurate identification of hidden metastases after lymph node dissection remains an important problem.Aim. тo evaluate the effect of histological examination characteristics and immunohistochemical measurement of pancytokeratin level on frequency of detection of papillary cancer hidden metastases in regional lymph nodes of the central zone in patients with clinical stage N0 papillary cancer.Materials and methods. The main group included 50 patients with stage ст1–2N0М0 primary papillary thyroid cancer. Dissected central lymph nodes of the patients prior to formalin fixation were extracted from the sample and inserted in individual paraffin blocks. Apart from standard histological examination, pancytokeratin level was measured immunohistochemically in the lymph nodes. The control group consisted of 200 patients for whom dissected central cell tissue was sectioned into blocks after formalin fixation. The number of lymph nodes in the dissected sample was measured by a pathomorphologist.Results. In the main group, the number of lymph nodes in the sample varied between 6 and 37 with mean of 20.7 ± 6.8; in the control group the number was lower: 3–25, mean 9.8 ± 5.1 (р = 0.000). In the main group, hidden metastases were detected more frequently than in the control group: in 30 (60 %) and 68 (34 %) cases, respectively (р = 0.001). In 20 (40 %) patients, immunohistochemical examination showed new metastases. use of this method allowed to detect 1 to 7 additional metastases (mean 2.4 ± 1.5 lymph node lesions).Conclusion. use of targeted dissection with extraction of lymph nodes led to significant increase in their numbers in the samples, and immunohistochemical examination allowed to detect a large number of hidden metastases in the central lymph nodes.

OGC P28 Raman Needle Probe Lymph Node Assessment for Oesophageal Cancer: The DOLOMITE Study

Abstract Background Oesophageal cancer (OC) accounts for 3% of all new cancer diagnosis in the UK. Presentation is often late, reflected in a poor 5-year survival rate of 12%. The importance of identifying lymph node (LN) metastases has been demonstrated as the single biggest prognostic factor. The current ‘gold standard’ diagnosis of LN metastases is from histopathological assessment of the tissue after surgical resection of the primary tumour and surrounding tissue. The use of imaging techniques to try and gain this information preoperatively is standard practice but far from perfect. Raman spectroscopy (RS) has been investigated as a diagnostic tool to detect cancer and pre-cancerous change in the oesophagus, and preliminary work demonstrating the application of vibrational spectroscopy (Raman and FTIR) to LN analysis has been undertaken. The development of Raman needle probes (RNP) with potential for use in-vivo furthers the clinical impact. The DOLOMITE study started recruitment in the summer of 2022 to investigate the ability of RNP to identify the presence of malignant deposits in resected lymph nodes. Methods Patients identified by the clinical team as needing oesophagectomy to treat their OC were invited to participate in the study. After the specimen has been resected, prior to formalin fixation, three lymph nodes are dissected representing gastric, para-oesophageal and sub-carinal nodes. These are bisected longitudinally with half remaining with the specimen. The half for research is snap frozen in liquid nitrogen until needed for analysis. Adjacent sections are cut from the nodes to create slides for Raman mapping and conventional H&E staining. The bulk node left is used for Raman probe analysis. Spectral data is then analysed using MATlab. Results At this early stage, our initial measurements have provided information used for calibration in advance of completing recruitment. The Raman mapping has been assessed for correlation with the pathology identified in the H&E slides taken from the lymph node block. This data in turn has provided the background for developing interpretation of the Raman probe data, with further samples we will use this to create a classification model. Conclusions With ongoing recruitment the study will be able to fully report by September 2023.

Effect of 80% ethanol or 10% formalin fixation, freezing at -20°C and staining on Myxobolus (Myxosporea) spores to be deposited in parasitological collections.

The preparation of myxosporeans for the description of myxospores and their preservation as type material in parasitological collections show great variations. Most frequently, formalin and ethanol are used for fixation and Giemsa solution for staining spores. In this work, authors studied the effect of 80% ethanol and 10% formalin fixation, freezing at -20°C and staining on the size and transparency of two Myxobolus species of cyprinid fishes, M. bramae and M. bliccae spore, and recommended a new method for the deposition of type material to parasitological collections in museums. The studies have commended that fresh spores from mature plasmodia are the best material for measuring the size and studying the inner structures, the number of polar tubules in polar capsules and the morphological characters of the intercapsular appendix. The obtained quantitative data suggest that cryo- and chemical preservation do not have a notable negative effect on spores compared to fresh samples but they decrease the transparency of spores. Staining the spores with Ziehl-Neelsen has proved to be a useful method for studying the fine structure without size reduction, while Giemsa staining induced a shrinkage of spores so it seems to be not ideal for description of a new species. When treating spores of Myxobolus spp. with Lugol's solution, iodinophilous vacuoles in the sporoplasm were not recognised but visualisation of the coils of polar tubules was enhanced. As a type material for newly described species, authors suggest phototypes and spores fixed in 80% ethanol to be deposited into collections, as this preservation method is suitable for subsequent research, such as re-measurements and molecular analysis.

Underestimation of endoscopic size in large gastric epithelial neoplasms.

Endoscopic submucosal dissection (ESD) is an effective method for resecting gastric adenomas and adenocarcinomas. A significant discrepancy was observed between endoscopic and pathological sizes in samples obtained from patients undergoing ESD. This study elucidates the factors affecting size discrepancy after formalin fixation. The records of 64 patients with 69 lesions were analyzed, including 50 adenomas and 19 adenocarcinomas. Data on location, gross shape, histology, and size after fixation in formalin were collected. The mean size of the resected specimen appeared to decrease after formalin fixation (37.5 mm prefixation vs. 35.8 mm postfixation, p<0.05). The mean long axis diameter of the lesions was 20.3±7.9 mm prefixation and 13.4±7.9 mm postfixation. Size differences in lesions smaller than 20 mm were significantly greater than those in lesions larger than 20 mm (7.6±5.6 mm vs. 2.5±5.8 mm, p<0.01). In multivariate analysis, a tumor size of ≥20 mm was found to be an independent factor affecting size postformalin fixation (p<0.05). The endoscopic size of lesions before ESD may be underestimated in tumors larger than 20 mm in size. Therefore, increased attention must be paid during ESD to avoid instances of incomplete resection.

Protocol for the Systematic Fixation, Circuit-Based Sampling, and Qualitative and Quantitative Neuropathological Analysis of Human Brain Tissue.

Human brain tissue has long been a critical resource for neuroanatomy and neuropathology, but with the advent of advanced imaging and molecular sequencing techniques, it has become possible to use human brain tissue to study, in great detail, the structural, molecular, and even functional underpinnings of human brain disease. In the century following the first description of Alzheimer's disease (AD), numerous technological advances applied to human tissue have enabled novel diagnostic approaches using diverse physical and molecular biomarkers, and many drug therapies have been tested in clinical trials (Schachter and Davis, Dialogues Clin Neurosci 2:91-100, 2000). The methods for brain procurement and tissue stabilization have remained somewhat consistently focused on formalin fixation and freezing. Although these methods have enabled research protocols of multiple modalities, new, more advanced technologies demand improved methodologies for the procurement, characterization, stabilization, and preparation of both normal and diseased human brain tissues. Here, we describe our current protocols for the procurement and characterization of fixed brain tissue, to enable systematic and precisely targeted diagnoses, and describe the novel, quantitative molecular, and neuroanatomical studies that broadly expand the use of formalin-fixed, paraffin-embedded (FFPE) tissue that will further our understanding of the mechanisms underlying human neuropathologies.

DIGE Saturation Labeling for Scarce Amounts of Protein from Formalin-Fixed Paraffin-Embedded (FFPE) Tissue.

In this chapter, we describe the utility of fluorescence two-dimensional difference gel electrophoresis (2D-DIGE) as a proteomics platform for the global detection of expressed proteins in formalin-fixed paraffin-embedded (FFPE) tissues and its use for biomarker discovery/identification of proteins that may contribute to cancer development and progression. Formalin fixation and paraffin embedding of tissue is the standard processing methodology practiced in pathology laboratories worldwide, resulting in a highly stable form of tissue that is easily stored due to its inherent stability at room temperature. Consequently, FFPE tissues represent an attractive reservoir of clinical material for conducting retrospective protein biomarker analysis. A limitation for proteomics research in this type of clinical sample is the amount of viable protein that can be obtained from fixed tissues. Tissue biopsies are precious samples that can generally be acquired in very small amounts due to the invasive nature of the sample collection, mainly during surgery or biopsy. Subsequently, the amount of extracted protein can be, in many cases, very limited. The saturation 2D-DIGE technology has emerged as a useful method for protein analysis where only scarce amounts of protein are available. This approach can be adapted successfully to label low-level protein isolated from FFPE tissue.

Abstract PR007: Three-dimensional genomic analysis of human pancreatic intraepithelial neoplasia (PanIN) reveals striking multifocality and genetic heterogeneity

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease with a 5 year survival rate of 11%. PDAC arises from precursor lesions, the most common of which is pancreatic intraepithelial neoplasia (PanIN). Molecular analysis of human PanINs is critical to understand early pancreatic tumorigenesis, which could inform risk stratification, early detection, and cancer prevention approaches. Because PanINs are microscopic, it is challenging to determine their full extent and anatomic relationships using two dimensional histological sections. To determine the density and connectivity of PanINs, we utilized a novel machine learning algorithm (CODA) for three-dimensional (3D) reconstruction and cellular quantification. Large blocks of grossly normal pancreas were harvested from 39 surgical pancreatectomy specimens, followed by formalin fixation, paraffin-embedding, and complete serial sectioning. 3D reconstruction using CODA revealed striking multifocality of PanINs within the pancreata of most analyzed patients, with more than 600 spatially PanINs being modeled. Next, to determine whether these multifocal PanINs arose as independent neoplasms or via intraductal spread of a single PanIN, we assessed their clonal relationships using somatic multi-region DNA next generation sequencing (NGS). To do so, 8 additional blocks of grossly normal pancreatic tissue were harvested and 3D modeled, yielding 37 anatomically distinct PanINs for genomic analysis. Each spatially unconnected PanIN was separately microdissected in five different regions to assess both intra-PanIN and inter-PanIN genetic heterogeneity. 99 samples from the 37 PanIN lesions underwent targeted NGS using a panel covering major drivers of pancreatic ductal neoplasia. For PanINs with sufficient DNA, whole exome sequencing and deep sequencing of KRAS was also performed. Across the 8 blocks, 10 PanINs shared no mutations with other PanINs in the same block, indicating independent clonal origin. In addition, 8 PanINs shared only KRAS hotspot mutations with numerous other unshared mutations, likely indicating independent PanINs that shared hotspot mutations by chance. Six spatially separate low grade PanINs shared both driver and passenger mutations with at least one other spatially unconnected PanIN, demonstrating the ability of PanIN cells to dissociate from one lesion and establish unconnected, genetically related PanINs nearby. Furthermore, 5 PanINs harbored multiple KRAS mutations within a single PanIN, suggesting a polyclonal origin. One PanIN lacked any driver gene mutations. The genetic origins of the remaining 7 spatially separate PanINs could not be resolved due to a lack of discrete mutations found by targeted sequencing. Our 3D genomic analysis of anatomically distinct PanINs demonstrates that the unexpectedly large number of PanINs in normal pancreas most often arise independently, providing new foundations for our understanding of early pancreatic tumorigenesis. Determining the mechanisms for this multifocal neoplasia is an important direction for future research. Citation Format: Alicia M. Braxton, Ashley Kiemen, Rachel Karchin, Yuchen Jiao, Pei-Hsun Wu, Ralph H Hruban, Denis Wirtz, Laura D. Wood. Three-dimensional genomic analysis of human pancreatic intraepithelial neoplasia (PanIN) reveals striking multifocality and genetic heterogeneity [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr PR007.

Morphological structure of rat tongue using light and scanning electron microscopy.

The rat is one of the most commonly used animals in biological research and experimental investigations in medicine. The ultrafine structural components of the epithelium differ depending on the anatomy of the animal and the papilla type. Animal adaptation to food types and environmental circumstances may also be linked to morphological diversity. In the current study, seven male Wistar rat tongues were investigated. For scanning electron microscope (SEM), two rat tongues were immersed in a 10% formalin solution and the other two rat tongues were immersed in a 2.5% glutaraldehyde solution. The tongues of three rats were fixed for regular histological evaluation using triple staining. The three primary components of the Wistar rat tongue are the apex, body and root. The apex had a rounded and bifurcated shape. Filiform papillae and gustatory papillae were easily identified on the dorsal side of the tongue. There were three forms of gustatory papillae; fungiform papillae, vallate papillae and foliate papillae. The purpose of this study was to expose the tongue morphology of the Wistar rat species, which is widely used in investigations. Also, we wanted to show that formalin fixation can be utilized for morphological research in SEM. Finally, the Wistar rat tongue was thoroughly investigated and compared to those of other species.

Abstract 230: Spinal Cord Injury In A 14 Days Survival Rat Model Of 6 And 8 Min Of Ventricular Fibrillation Cardiac Arrest, Resuscitated With Conventional Chest Compression

Introduction: Impact of global ischemia and reperfusion on brain in resuscitation animal models is well investigated. Investigations on vulnerability of spinal cord in such models are rarely reported. We investigated the spinal cord in a cardiopulmonary resuscitation ventricular fibrillation cardiac arrest (VFCA) rat model and hypothesized that an untreated CA of 6 and 8 min will lead to histological damage and dysfunction in the resuscitated animal′s spinal cord, depending on CA duration. Methods: Adult male Sprague-Dawley rats (350-450 g) were subjected to 6 (n=8) or 8 min (n=14) of VFCA and resuscitated with conventional chest compressions with a thumper device, mechanical ventilation and drugs (epinephrine, bicarbonate, heparin). After defibrillation and ROSC, rats survived for 14 days. The spinal cord was removed from the spine after fixation in formalin and embedded in paraffin. Transverse sections of cervical, thoracic and lumbar spinal cord were stained with hematoxylin and eosin (HE). Immunohistochemistry was performed with antibodies against GFAP for astrocytes and Iba1 for microglia. Sections were evaluated by a pathologist blinded to treatment regimens, and compared to sham animals (n=5). Results: Resuscitated animals showed high mortality; after 6 min CA four animals and after 8 min CA eleven animals died before endpoint. Surviving animals showed focal bilateral symmetrical loss of neurons and distinct gliosis in the gray matter of the thoracic spinal cord level with the central canal. Similar lesions were detected in other segments of the spinal cord, but were located predominantly in the ventral horn of the spinal cord. These lesions were accompanied by dilated myelin sheaths in the white matter. Immunohistochemistry showed activation of astrocytes and microglia in the damaged regions. There were no differences detectable between animals subjected to 6 or 8 min VFCA. Sham animals showed no lesions in the spinal cord. Conclusions: We found consistent histological damage in the spinal cords of rats resuscitated after 6 and 8 min untreated VFCA with CPR after 14 days survival but without clinical symptoms in these animals. However, similar lesions might be cause of paralysis and urinary retention found in animals that died before endpoint.

Effect of formalin fixation on size and weight of Atlantic wolffish (Anarhichas lupus) and spotted wolffish (Anarhichas minor) oocytes

Oocytes were sampled from Atlantic wolffish (Anarhichas lupus) and spotted wolffish (Anarhichas minor) to investigate the effect of fixation and storage in formalin on oocyte size and weight over time. The effect of formalin on oocyte size was dependent on the original size of the oocyte. As the effect of fixation was similar for the two species, a common formula for the conversion of fixed oocyte size/weight to fresh oocyte size/weight was developed. The relationships were negative and nonlinear, the smallest oocytes increased ∼11 % in diameter and ∼15 % in weight when fixed in formalin, whereas the largest oocytes decreased about 4 % in diameter and 7 % in weight. After fixation, oocyte diameter was stable for up to 2, but not 10 years and for up to 10 years for oocytes at CA and VIT stage, respectively.

Enzyme-labeled Antigen Method: Factors Influencing the Deterioration of Antigen-binding Activity of Specific Antibodies during Formalin Fixation and Paraffin Embedding.

The enzyme-labeled antigen method is an immunohistochemical technique detecting plasma cells producing specific antibodies in tissue sections. The probe is an antigen labeled with an enzyme or biotin. This immunohistochemical technique is appliable to frozen sections of paraformaldehyde (PFA)-fixed tissues, but it has been difficult to apply it to formalin-fixed, paraffin-embedded (FFPE) sections. In the current study, factors inactivating the antibody reactivity during the process of preparing FFPE sections were investigated. Lymph nodes of rats immunized with horseradish peroxidase (HRP) or a mixture of keyhole limpet hemocyanin/ovalbumin/bovine serum albumin were employed as experimental models. Plasma cells producing specific antibodies, visualized with HRP (as an antigen with enzymatic activity) or biotinylated proteins in 4% PFA-fixed frozen sections, significantly decreased in unbuffered 10% formalin-fixed frozen sections. The positive cells were further decreased by paraffin embedding following formalin fixation. In paraffin-embedded sections fixed in precipitating fixatives such as ethanol and acetone and those prepared with the AMeX method, the antigen-binding reactivity of antibodies was preserved. Fixation in periodate-lysine-paraformaldehyde and Zamboni solution also kept the antigen-binding reactivity in paraffin to some extent. In conclusion, formalin fixation and paraffin embedding were major causes inactivating antibodies. Precipitating fixatives could retain the antigen-binding reactivity of antibodies in paraffin-embedded sections.

Evaluation of Formalin-Fixed and FFPE Tissues for Spatially Resolved Metabolomics and Drug Distribution Studies.

Fixation of samples is broadly used prior to the histological evaluation of tissue samples. Though recent reports demonstrated the ability to use fixed tissues for mass spectrometry imaging (MSI) based proteomics, glycomics and tumor classification studies, to date comprehensive evaluation of fixation-related effects for spatially resolved metabolomics and drug disposition studies is still missing. In this study we used matrix assisted laser desorption/ionization (MALDI) and desorption electrospray ionization (DESI) MSI to investigate the effect of formalin-fixation and formalin-fixation combined with paraffin embedding on the detectable metabolome including xenobiotics. Formalin fixation was found to cause significant washout of polar molecular species, including inorganic salts, amino acids, organic acids and carnitine species, oxidation of endogenous lipids and formation of reaction products between lipids and fixative ingredients. The slow fixation kinetics under ambient conditions resulted in increased lipid hydrolysis in the tissue core, correlating with the time-dependent progression of the fixation. Paraffin embedding resulted in subsequent partial removal of structural lipids resulting in the distortion of the elucidated biodistributions.

Volumetric assessment and longitudinal changes of subcortical structures in formalinized Beagle brains.

High field MRI is an advanced technique for diagnostic and research purposes on animal models, such as the Beagle dog. In this context, studies on neuroscience applications, e.g. aging and neuro-pathologies, are currently increasing. This led to a need for reference values, in terms of volumetric assessment, for the structures typically involved. Nowadays, several canine brain MRI atlases have been provided. However, no reports are available regarding the measurements’ reproducibility and little is known about the effect of formalin on MRI segmentation. Here, we assessed the segmentation variability of selected structures among operators (two operators segmented the same data) in a sample of 11 Beagle dogs. Then, we analyzed, for one Beagle dog, the longitudinal volumetric changes of these structures. We considered four conditions: in vivo, post mortem (after euthanasia), ex vivo (brain extracted and studied after 1 month in formalin, and after 12 months). The MRI data were collected with a 3 T scanner. Our findings suggest that the segmentation procedure was overall reproducible since only slight statistical differences were detected. In the post mortem/ ex vivo comparison, most structures showed a higher contrast, thereby leading to greater reproducibility between operators. We observed a net increase in the volume of the studied structures. This could be justified by the intrinsic relaxation time changes observed because of the formalin fixation. This led to an improvement in brain structure visualization and segmentation. To conclude, MRI-based segmentation seems to be a useful and accurate tool that allows longitudinal studies on formalin-fixed brains.

Adapted lead (Pb) histochemical detection procedure in tissue samples of blue crabs Callinectes danae (Smith, 1869)

Lead (Pb) cellular deposits can be detected by histochemistry using a classical potassium dichromate - formalin fixation and post-fixation with 3% potassium dichromate solution. Here we describe an alternative procedure for Pb histochemical detection in the blue crab Callinectes danae. Animals were exposed to Pb (2 µg/mL) for 24, 48 and 96h. Then, buffered formalin solution was used for hepatopancreas and gills fixation, with tissue sections incubated in 3% potassium dichromate at 40 °C for seven days. Counterstains were tested with better results reached using 0.5% phloxine and 1% methylene blue dyes. This adapted method favors Pb deposition studies in wildlife animals since it uses ordinary steps for tissue collection and standard histological procedures. Besides, this method is cheaper and simpler than the classical one for lead deposits detection.

Effect of formalin fixation on tumour size and margins in head and neck cancer specimens.

To quantify tumour size and margin shrinkage due to formalin fixation in head and neck cancer specimens and determine its effect on tumour staging and margin clearance. Tumour specimens were measured immediate post-resection and at 24 hours after fixation. Tumour was measured in 2 dimensions and one margin was measured. Shrinkage was categorised as < 10%, 10-20%, and > 20%. Effect of shrinkage on tumour stage and margin clearance were evaluated. A total of 50 specimens were analysed. The tumour AP (Anterior Posterior) and SI (Superior Inferior) measurements showed significant shrinkage with a mean difference of 22.93% and 21.69% respectively. > 20% shrinkage was noted in 78% of AP and 58% of SI measurements. Margins showed a mean difference of 25.61%. 84% of margins demonstrated > 20% shrinkage. In all, 46.7% of T3 and 23% of T2 tumours pre-fixation were downstaged to T2 and T1, respectively, post-fixation. Formalin fixation alone can be responsible for significant shrinkage of tumour and margin dimensions in head and neck specimens. It is suggested that decisions regarding the treatment plan should be made on clinical staging of primary tumour rather than pathological staging. In addition, post-excision pre-fixation margins should be considered for treatment planning.

Formalin Fixation, Delay to Fixation, and Time in Fixative Adversely Impact Copy Number Variation Analysis by aCGH.

Although molecular profiling of DNA isolated from formalin-fixed, paraffin-embedded (FFPE) tumor specimens has become more common in recent years, it remains unclear how discrete FFPE processing variables may affect detection of copy number variation (CNV). To better understand such effects, array comparative genomic hybridization (aCGH) profiles of FFPE renal cell carcinoma specimens that experienced different delays to fixation (DTFs; 1, 2, 3, and 12 hours) and times in fixative (TIFs; 6, 12, 23, and 72 hours) were compared to snap-frozen tumor and blood specimens from the same patients. A greater number of regions containing CNVs relative to commercial reference DNA were detected in DNA from FFPE tumor specimens than snap-frozen tumor specimens even though they originated from the same tumor blocks. Extended DTF and TIF affected the number of DNA segments with a copy number status that differed between FFPE and frozen tumor specimens; a DTF ≥3 hours led to more segments, while a TIF of 72 hours led to fewer segments. Importantly, effects were not random as a higher guanine-cytosine (GC) content and/or a higher percentage of repeats were observed among stable regions. While limiting aCGH analysis to FFPE specimens with a DTF <3 hours and a TIF <72 hours may circumvent some effects, results from FFPE specimens should be validated against fresh or frozen specimens whenever possible.

OA32 Minimally invasive, ultrasound-guided tissue biopsies of synovial tissue in children with Juvenile Idiopathic Arthritis for research: a feasibility study

Introduction/BackgroundWhen investigating disease mechanisms, site-specific differences in immune cell phenotype and function have highlighted the need to analyse cellular and molecular mechanisms at the tissue site directly. In adults, the ability to obtain synovial tissue biopsies using ultrasound-guided techniques, combined with advanced tissue analytics, has revolutionised our understanding of the cellular ecosystem that operates within the joint and how it contributes to disease. However, a similar approach in paediatric disease is lacking. Description/MethodAims: 1) To describe the protocol for undertaking minimally invasive ultrasound-guided synovial tissue biopsies in children and young people with arthritis, for the purpose of research, alongside routine clinical care. 2) To investigate whether high-quality synovial tissue can be obtained that is suitable for downstream applications including single cell profiling technologies, histology and digital spatial profiling.Treatment-naïve children with a diagnosis of Juvenile Idiopathic Arthritis, who were being referred for a corticosteroid joint injection were recruited from two large UK Paediatric Rheumatology centres. We established a workflow pipeline for performing synovial tissue biopsies in child and young people with arthritis, using standardised procedures for biopsy and sample processing. Procedures were performed by experienced paediatric interventional radiologists with experience of joint biopsy for diagnostic purposes. Following a general anaesthetic, required as part of routine clinical care and the establishment of sterility, synovial fluid was aspirated. Needle-biopsies were undertaken from the same needle insertion site and subsequently corticosteroid was injected into the joint. Thickened synovium was graded via ultrasonography. Participating families completed questionnaires prior to and following synovial biopsy.Discussion/Results11 participants were recruited to the study over a nine month period, with a median age of 7 years (range 1-16 years); 91% were female. Samples obtained included core synovial biopsies, paired synovial fluid and peripheral blood. Synovial tissue fragments were processed for histology by formalin fixation and cryopreserved for downstream applications, including RNA sequencing and cell culture. Quality control indices included histological analysis to ensure the biopsied material was characteristically synovium and to grade the severity of inflammation. No significant complications were reported; however, one child had a mild haemarthrosis controlled with cold saline wash out and cold compresses.Key learning points/ConclusionObtaining biopsies of synovial tissue in children with Juvenile Idiopathic Arthritis for the purpose of research, alongside clinical care is feasible. Analysis of tissue direct from the site of inflammation with single-cell RNA sequencing in children is achievable.

P454 Massive parallel fungal sequencing on formalin-fixed tissues: development and contribution in integrated histomolecular diagnosis

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PM ObjectivesHistopathology is the gold standard for distinguishing between colonization and fungal infection, but it does not provide a precise diagnosis of genera/species. However, if the culture is negative or if no specimen is sent to the Mycology laboratory, only the specimen sent to the Pathology department is available. Formalin fixation and paraffin embedding (FFPE) cause DNA damage, making it difficult to perform molecular techniques.The objective was to develop and evaluate the contribution of massive parallel sequencing (MPS) to FFPE tissues.MethodsHistopathological review of all cases was performed. Then, DNA extraction from FFPE tissues was optimized by: (1) macrodissecting the fungal-rich area on the paraffin block; (2) comparing the efficiency of two DNA extraction kits (QIAamp DNAmicro-kit, QIAGEN; Maxwell 16 LEVRNA FFPE Purification kit, Promega, optimized for RNA and DNA extraction), by comparison of Aspergillus fumigatus and Mucorale specific PCR results for 30 cases. For 124 other cases, the sensitivity of two primer pairs (ITS3/4 and MITS2A/2B) was tested for identification by Sanger sequencing and then MPS. Finally, a histomolecular comparison was performed. This work was funded by the Société de Pathologie Infectieuse de Langue Française (SPILF).ResultsTo optimize extraction, DNA was extracted by both kits from samples of 16 mucormycoses and 14 A. fumigatus infections. PCR sensitivity was better with the QIAGEN extraction kit [100% (30/30)] compared to the Promega kit [86.7% (26/30)].PCR amplification of fungal DNA from an additional 124 FFPE samples was performed. The primer pairs ITS3/4 and MITS2A/2B, allowed: (1) identification by Sanger sequencing-histopathological analysis in 38.7% (48/124) of the cases in total, and more specifically 33% (41/124) of cases with the ITS3/4 primers and 32.3% (40/124) of cases with the MITS2A/B primers; and (2) identification by integrated SMP-histopathological analysis in 75% (93/124) of all cases (primers ITS3/4 and MITS2A/2B), and more specifically 66.1% (82/124) for ITS3/4 and 62.1% (77/124) for MITS2A/B (both primer pairs did not detect/amplify the same fungal genera/species). The combination of all results from Sanger sequencing and MPS led to fungal identification in 75.8% (94/124) of cases. In total, the addition of NGS to Sanger sequencing increased the diagnostic proportion by 36.3% (45/124; P <.0001). Example of integrated histomolecular diagnosis (Fig. 1): patient with a pseudotumor presentation of pulmonary invasive aspergillosis (A: thoracic CT scan; B: macroscopic examination of lesion after formalin fixation; C, D, E: Hematoxylin-eosin-safran, x20 and x400: observation of a necrotic mass caused by hyalohyphomycetes; F: Grocott, x400) with no culture or molecular identification available on fresh tissue. In contrast, identification by MPS on FFPE tissue was compatible with morphological analysis: Aspergillus section Fumigati, leading to the integrated histomolecular diagnosis of pulmonary invasive aspergillosis.ConclusionThe development of the fungal MPS on FFPE tissues is innovative and unprecedented for the achievement of an integrated histomolecular diagnosis in fungal pathology. It increases significantly the diagnostic proportion by 36.3%. This strategy can be used in hospitals and could improve patient management, especially when no sample is sent to the Mycology laboratory or when the culture is negative.

Measurement of distal intramural spread and the optimal distal resection by naked eyes after neoadjuvant radiation for rectal cancers

BackgroundThe safe distance between the intraoperative resection line and the visible margin of the distal rectal tumor after preoperative radiotherapy is unclear. We aimed to investigate the furthest tumor intramural spread distance in fresh tissue to determine a safe distal intraoperative resection margin length.MethodsTwenty rectal cancer specimens were collected after preoperative radiotherapy. Tumor intramural spread distances were defined as the distance between the tumor’s visible and microscopic margins. Visible tumor margins in fresh specimens were identified during the operation and were labeled with 5 - 0 sutures under the naked eye at the distal 5, 6, and 7 o’clock directions of visible margins immediately after removal of the tumor. After fixation with formalin, the sutures were injected with nanocarbon particles. Longitudinal tissues were collected along three labels and stained with hematoxylin and eosin. The spread distance after formalin fixation was measured between the furthest intramural spread of tumor cells and the nanocarbon under a microscope. A positive intramural spread distance indicated that the furthest tumor cell was distal to the nanocarbon, and a negative value indicated that the tumor cell was proximal to the nanocarbon. The tumor intramural spread distance in fresh tissue during the operation was 1.75 times the tumor intramural spread distance after formalin fixation according to the literature.ResultsAt the distal 5, 6, and 7 o’clock direction, seven (35%), five (25%), and six (30%) patients, respectively, had distal tumor cell intramural spread distance > 0 mm. The mean and 95% confidence interval of tumor cell intramural spread distance in fresh tissue during operation was − 0.3 (95%CI − 4.0 ~ 3.4) mm, − 0.9 (95%CI − 3.4 ~ 1.7) mm, and − 0.4 (95%CI − 3.5 ~ 2.8) mm, respectively. The maximal intraoperative intramural spread distances in fresh tissue were 8.8, 7, and 7 mm, respectively.ConclusionsThe intraoperative distance between the distal resection line and the visible margin of the rectal tumor after radiotherapy should not be less than 1 cm to ensure oncological safety.

Comparison of pre-formalin fixation and postfixation temporal artery biopsy lengths

ObjectifComparer la longueur de la biopsie de l'artère temporale avant et après la fixation dans la formaline et noter les corrélations avec le diagnostic histopathologique. NatureÉtude prospective portant sur une série de cas. ParticipantsPatients qui ont subi une biopsie de l'artère temporale entre juin 2020 et octobre 2021. MéthodesA été comparée la longueur de la biopsie avant et après la fixation dans la formaline. Le paramètre principal était la différence au chapitre de la longueur de l'artère temporale mesurée avant la fixation par le chirurgien comparativement à la mesure après la fixation par l'anatomopathologiste. RésultatsOn a inclus 47 biopsies consécutives de 46 patients (1 patient a dû subir une deuxième biopsie). L’âge moyen des patients était de 75,3 ± 8,4 ans (fourchette : 49–94 ans); 74 % des patients (34 sur 46) étaient des femmes. La longueur moyenne de la biopsie avant la fixation s’élevait à 2,36 ± 0,58 cm (fourchette : 1,1–4,5 cm). La longueur moyenne de la biopsie après la fixation était de 2,09 ± 0,59 cm (fourchette : 0,6–3,8 cm). La différence moyenne (soit le raccourcissement après la fixation) était de 0,27 ± 0,24 cm (p = 0,0298), et 36 % des biopsies (17 sur 47) étaient positives. On ne note aucune différence significative entre la longueur de la biopsie de l'artère temporale avant la fixation (p = 0,38) ou le raccourcissement après la fixation (p = 0,24) selon que la biopsie ait été positive ou négative. D'après l'analyse univariée, l’élévation de la vitesse de sédimentation se chiffrait à 31,3 mm/h (fourchette : 4–88 mm/h) dans les biopsies négatives, comparativement à 54,5 mm/h (fourchette : 29–98 mm/h) dans les biopsies positives (p = 0,01); le taux de protéine C-réactive s’élevait à 17,4 mg/L (fourchette : 0,2–145 mg/L) dans les biopsies négatives, comparativement à 78,56 mg/L (fourchette : 5–244,4 mg/L) dans les biopsies positives (p = 0,003), et la numération plaquettaire était de 254,9 × 109/L (fourchette : 134–570 × 109/L) dans les biopsies négatives, comparativement à 393,8 × 109/L (fourchette : 210–593 × 109/L) dans les biopsies positives (p < 0,001). Toutes ces composantes étaient associées à un diagnostic anatomopathologique positif. ConclusionsEn moyenne, la biopsie de l'artère temporale était plus courte d'environ 0,27 cm selon les rapports d'anatomopathologie, comparativement à la mesure réalisée avant la fixation. Les chirurgiens doivent tenir compte de ce raccourcissement en incluant une marge de sécurité d'au moins 0,3 cm, de sorte qu'ils excisent au moins 2,3 cm s'ils souhaitent obtenir une longueur de 2 cm après la fixation.