Formalin Fixation Research Articles

The impact of formalin fixation in the elemental content of tissues: Parametrization up to 48 h

Formalin fixation is a crucial step in the preparation of tissue samples for anatomic pathology studies, however, research showed that prolonged formalin fixation, can alter the elemental composition of tissues with implications for pathology and biomedical research. In this study we aim to assess and parametrize the influence of short-term formalin fixation in tissues.Six sets of human colon tissue samples were exposed to different formalin fixation times, up until 48 h. The elemental content throughout time was compared to the elemental content of the snap-frozen sample of the same tissue that was not exposed to formalin using Energy Dispersive X-ray Fluorescence.Results showed a clear decrease of Cl and K concentration in the tissues solution reaching a plateau between 1 h and 3 h of fixation. Also, there is an uptake of P in the tissue, likely due to the buffered formalin solution, within the first 15 min of fixation. This behaviour was concomitant with an increase of Na determined using Particle Induced gamma-ray emission analysis (PIGE) and Elastic Backscattering Spectroscopy (EBS). The dynamic changes determined in the studied elements demonstrate that a better understanding of the fixation-related mechanisms is in demand as well as optimization of the fixation protocols implemented in anatomic pathology laboratories.

Effects of Extended Fixation on Advanced Gastric Cancer HER2 Status Assessment Using IHC and FISH.

In gastric cancer, accurate determination of human epidermal growth factor receptor type 2 (HER2) status is crucial for treatment decision-making. However, the optimal formalin fixation time of gastric cancer specimens for HER2 status determination remains unestablished. Here, we investigated real-world data on formalin overfixation and its effect on HER2 status determination in gastric cancer. We comprehensively analyzed HER2 testing results in 228 gastric cancer specimens, including those subjected to formalin overfixation. Subsequently, we divided 52 resected specimens of advanced gastric cancer into three groups and studied the effects of short-term (6-72 h) and long-term (1 and 2 weeks) fixation on HER2 status determination using immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). A total of 21.5% (49/228) of the specimens were HER2-positive, whereas 78.5% (179/228) were negative. Among the HER2-negative specimens, no biopsies were overfixed, whereas 12.5% (9/72) of the surgical resection specimens were overfixed. The HER2 status of the 6-72-h group was 82.7% and 76.9% identical to that of the 1- and 2-week groups, when determined using IHC, and 73.1% and 36.5%, when determined using FISH, respectively. However, HER2 determination was not feasible in 26.9% and 63.5% of the specimens in the 1- and 2-week groups, respectively. Formalin overfixation may hinder the determination of HER2 status and should be avoided in gastric cancer sample preparation.

Neuropathological Applications of Microscopy with Ultraviolet Surface Excitation (MUSE): A Concordance Study of Human Primary and Metastatic Brain Tumors.

Whereas traditional histology and light microscopy require multiple steps of formalin fixation, paraffin embedding, and sectioning to generate images for pathologic diagnosis, Microscopy using Ultraviolet Surface Excitation (MUSE) operates through UV excitation on the cut surface of tissue, generating images of high resolution without the need to fix or section tissue and allowing for potential use for downstream molecular tests. Here, we present the first study of the use and suitability of MUSE microscopy for neuropathological samples. MUSE images were generated from surgical biopsy samples of primary and metastatic brain tumor biopsy samples (n = 27), and blinded assessments of diagnoses, tumor grades, and cellular features were compared to corresponding hematoxylin and eosin (H&E) images. A set of MUSE-treated samples subsequently underwent exome and targeted sequencing, and quality metrics were compared to those from fresh frozen specimens. Diagnostic accuracy was relatively high, and DNA and RNA integrity appeared to be preserved for this cohort. This suggests that MUSE may be a reliable method of generating high-quality diagnostic-grade histologic images for neuropathology on a rapid and sample-sparing basis and for subsequent molecular analysis of DNA and RNA.

Effects of autolysis and prolonged formalin fixation on histomorphology and immunohistochemistry of normal canine brain tissue: an experimental study.

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.

Forensic pilot application of rehydrating solutions on human cadaveric skin: what are the effects on hemorrhagic infiltrates?

The microscopic evaluation of hemorrhagic infiltrates is crucial in forensic diagnostics, but it proves challenging in corificated and mummified cadavers. In these cases, pre-treatment with rehydrating solutions is recommended, although their effects on the hemorrhagic infiltrate are not well understood. In this pilot study, we microscopically investigated the effect of two different rehydrating solutions-Sandison's solution and fabric softener-on well-preserved human cadaveric skin samples taken from areas affected by an ecchymotic lesion, comparing them with direct fixation in formalin. Specifically, we examined the topographic distribution of the hemorrhagic infiltrate in each layer of the skin by assigning a semi-quantitative score, conducted mutual comparisons, and performed statistical analysis. Histologically, compared to direct fixation in formalin, a slight and statistically non-significant reduction in the hemorrhagic infiltrate was observed in samples pre-treated with fabric softener. On the other hand, a more pronounced and statistically significant decrease in scores was observed in samples pre-treated with Sandison's solution. This effect is likely due to the fact that Sandison's solution, due to its components, exerts an osmotic effect, partially inducing osmotic lysis of red blood cells. Overall, extensive areas of hemorrhagic infiltrates were preserved, although to a lesser extent, while smaller foci were markedly reduced, sometimes even disappearing. The findings suggest that Sandison's solution has a detrimental effect on cutaneous hemorrhagic infiltrates, emphasizing the importance of being cautious and conducting dual sampling, using both formalin and a rehydrating solution, for forensic examination of mummified or corificated skin samples.

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

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

A Fast-Tracking Sample Preparation Protocol for Proteomics of Formalin-Fixed Paraffin-Embedded Tumor Tissues.

Archived tumor specimens are routinely preserved by formalin fixation and paraffin embedding. Despite the conventional wisdom that proteomics might be ineffective due to the cross-linking and pre-analytical variables, these samples have utility for both discovery and targeted proteomics. Building on this capability, proteomics approaches can be used to maximize our understanding of cancer biology and clinical relevance by studying preserved tumor tissues annotated with the patients' medical histories. Proteomics of formalin-fixed paraffin-embedded (FFPE) tissues also integrates with histological evaluation and molecular pathology strategies, so that additional collection of research biopsies or resected tumor aliquots is not needed. The acquisition of data from the same tumor sample also overcomes concerns about biological variation between samples due to intratumoral heterogeneity. However, the protein extraction and proteomics sample preparation from FFPE samples can be onerous, particularly for small (i.e., limited or precious) samples. Therefore, we provide a protocol for a recently introduced kit-based EasyPep method with benchmarking against a modified version of the well-established filter-aided sample preparation strategy using laser-capture microdissected lung adenocarcinoma tissues from a genetically engineered mouse model. This model system allows control over the tumor preparation and pre-analytical variables while also supporting the development of methods for spatial proteomics to examine intratumoral heterogeneity. Data are posted in ProteomeXchange (PXD045879).

Histomorphological Comparison of Tissues Fixed in Conventional Formalin and Eco-friendly Jaggery Solution: A Cross-sectional Study

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

Histopathological spectrum of lung findings in autopsy cases

Background: The incidence of lung pathology is the most common autopsy finding and is responsible for a great deal of mortality and morbidity. It is affected by a wide variety of respiratory pathogens and is involved in many systemic diseases. It is secondarily involved in almost all forms of terminal disease. Radiological findings are usually nonspecific in lung diseases and need prompt histopathological examination to find out the exact cause of death. Aims and Objectives: The aim is to study the spectrum of histopathological findings in lung in autopsy cases. Materials and Methods: This is a retrospective study done in the Department of Pathology at Government Kilpauk Medical College and Hospital during the year 2022. Lung specimens were received from the Forensic medicine department. Gross findings were noted. Following adequate formalin fixation, the tissue specimens were processed and paraffin sectioning was done followed by hematoxylin and eosin staining. The findings were documented and the results were analyzed. Results: Among 100 cases studied during year maximum cases were seen in the age group of 31–40 years (32%). Males (72%) were more commonly affected than females (28%). The most common findings are Pulmonary edema (34%) followed by congestion (32%) and other findings include emphysematous change (13%), pneumonia (10%), tuberculosis (6%), metastatic adenocarcinomatous deposits (1%), primary well-differentiated adenocarcinoma (1%), pulmonary chondroma (1%), and bronchiectasis (1%). Conclusion: The most common finding observed was pulmonary edema. In our study, male preponderance was noted. This study emphasizes to scrutinize the histopathological findings in the lung, especially following a sudden death.

Methods of cadaver preservation used in Sri Lankan medical faculties; Merits and demerits

Anatomy is a fundamental education for healthcare professionals in understanding the structure and function of the human body. Cadavers - human bodies donated for scientific study - have been crucial for students and researchers to examine the three-dimensional structures and relationships within the body that cannot be taught by textbooks or digital models alone. Several techniques are employed to preserve cadavers. This article focuses on formalin fixation and phenoxy ethanol glycerine, the two most common practices used in Sri Lankan medical schools. This helps anatomy educators and embalmers to choose the right procedure with minimal exposure to hazardous chemicals.

Outcome of Adjuvant Radiotherapy and Adjuvant Chemoradiation for Oral Cancers with Close Margins of Resection.

Oral cancers comprise 50% of all head and neck malignancies in India which can be attributed to tobacco chewing. Advanced oral cancers are managed with surgery followed by adjuvant radiotherapy or adjuvant chemoradiation. There is paucity of studies regarding need for adjuvant treatment in oral cancers with close margins i.e. ≤ 0.5cm after formalin fixation. This study aims at documenting the oncological outcome with regards to loco-regional control, disease-free survival, overall survival and complications of adjuvant radiotherapy and adjuvant chemoradiation in operated oral cancer patients having close margins of resection. In this Retrospective cohort study, 163 patients with stage T1-T4 oral cancers operated between 2015 and 2019 who have fulfilled the inclusion and exclusion criteria, received adjuvant treatment and could be followed up for at least one year were included. At the end of 45months, the overall survival was 74.7% and disease specific survival was 82.7%. Among the 7 patients who defaulted radiotherapy, 4 patients succumbed to the disease. Complications were bone marrow depression (2 patients), dysphagia (17) and Trismus (1). Adjuvant radiotherapy should be given in oral cancer patients with close margins of resection since it improves the oncological outcome and disease specific survival and the benefit often outweighs the risk.

Quality assessment of histopathological stainings on prolonged formalin fixed thrombus tissues retrieved by mechanical thrombectomy.

Formalin-fixed retrieved clots from mechanical thrombectomy (MT) are now routinely studied using both conventional histopathologic techniques and immunohistochemistry (IHC). However, the effects of prolonged formalin fixation on the histological results of clot analysis remain unknown. The objective of this study was to investigate the effects of prolonged formalin fixation on quality of histopathologic stainings of thrombus tissues retrieved by MT. As part of the multicenter EXCELLENT registry, a total of 80 clots extracted by MT from acute ischemic stroke patients were randomly selected from the tissue database and assigned into four groups according to 10% neutral buffered formalin (NBF) fixation duration (1-30, 30-60, 60-90, and 90+ days, up to 2 years). Samples underwent processing and sectioning. Two serial sections for each case were stained with hematoxylin and eosin (H&E), Martius Scarlet Blue (MSB), and IHC for CD42b (platelet marker). An expert pathologist, who was blinded to tissue fixation duration and patient clinical data, assessed the quality of each stain including stainability, sensitivity, specificity, and consistency of stainings. No significant issues were encountered during tissue processing and sectioning. On H&E stain, 97.5% (78/80) of slides showed good-quality staining, demonstrating clear histological properties of the thrombus tissue as red blood cells (RBC) stained in red, fibrin/platelet stained in pink, and nuclei stained in blue with intranuclear detail. The same histological features were also successfully demonstrated on MSB for all 80 samples. One of the 80 samples (1.2%) showed that RBC lost stainability on H&E due to tissue autolysis. Clear positive signal of platelet staining was expressed in 98.8% of the samples (79/80) with minimal background staining on IHC. There was no significant difference in staining quality across different formalin fixation groups. A good quality of histopathological staining is achievable for the thrombus tissue fixed in 10% neutral buffered formalin for up to 2 years. The findings are limited to the thrombus tissue retrieved by MT and specific fixation and staining protocols used in the study. To apply these results to other tissue or experimental setups, further studies and validations would be necessary. This study was conducted as part of the EXCELLENT study: www.clinicaltrials.gov, unique identifier: NCT03685578.

Delineation of the healthy rabbit tongue by immunohistochemistry – A technical note

In the oral cavity the tongue is an important muscular organ that supports the swallowing of food and liquids. It is responsible for the sense of taste, based on the many different taste buds it contains. Research in the field of tongue diseases demands for suitable preclinical models. The healthy rabbit tongue may therefore serve as baseline and reference for the pathological situation. With this consideration, we covered the fixation and histological stainings as well as the immunohistochemical labelling of the healthy rabbit tongue. In this technical note, initial choice of the fixative is discussed, with a comparison of formalin fixation and subsequent paraffin embedding versus cryopreservation. Moreover, we delineate the effect of an antigen retrieval step for formalin fixation by several examples. Finally, we provide ECM markers collagen I, collagen III, fibronectin, α-SMA and elastin staining as well as ki67 for proliferative status and PAR-2 protein expression as a marker for inflammatory status and nociception in tongue sections, mainly from the tongue body. Technically, we found superiority of paraffin sections for collagen I, collagen III, fibronectin, ki67 and α-SMA labelling, for selected detections systems. As for ECM components, the lamina propria was very rich in collagen and fibronectin, while the muscular body of the tongue showed only collagen and fibronectin positive areas between the muscle fibers. Moreover, α-SMA was clearly expressed in the walls of arteries and veins. The inflammatory marker PAR-2 on the other hand was prominently expressed in the salivary glands and to some extent in the walls of the vessels. Particular PAR-2 expression was found in the excretory ducts of the tongue. This technical note has the aim to provide baseline images that can be used to compare the pathological state of the diseased rabbit tongue as well as for inter-species comparison, such as mouse or rat tongue. Finally, it can be used for the comparison with the human situation.

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

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

Cryo-X-ray phase contrast imaging enables whole biopsy imaging prior to multi-omic analysis

Abstract Background Analysis of myocardial biopsies using conventional two-dimensional (2D) histological techniques is limited in the detection of important three-dimensional (3D) structural features, such as disarray, due to sectioning and artefacts from dehydration or chemical processing. More advanced techniques such as light-sheet microscopy, micro-computed tomography or synchrotron radiation-based X-ray phase contrast imaging can provide non-destructive, 3D histological information at high resolution for formalin-fixed and/or paraffin-embedded (FFPE) biopsies. However, formalin fixation induces crosslinking of proteins which can impact quality and their subsequent analysis, for example, fragmentation of nucleic acids. A method for imaging 3D structure of myocardial biopsies in near-native state is needed which can then be followed by downstream multi-omic applications. Purpose To develop a technique called cryogenic X-ray phase contrast imaging (Cryo-X-PCI) to image snap-frozen myocardial biopsies at controlled low temperatures, followed by multi-omic analysis starting with analysis of DNA and RNA. Methods Myocardial biopsies (n=46) were obtained from C57BL/6 mice and snap-frozen in liquid nitrogen fresh or after 4% paraformaldehyde (PFA);10% formalin fixation. Pilot human aortic stenosis biopsies were also included (n=3, fresh). X-PCI was performed at -70°C using a cryojet, 20 keV beam energy and 0.65 µm detector pixel size (Figure 1). Datasets were reconstructed using Gridrec algorithm and Paganin phase retrieval. Morphologic analysis (myocardial disarray index, fractional anisotropy, helical angle, and intrusion angle) of biopsies was performed using a structure tensor-based approach via in-house MATLAB script. After imaging, DNA and RNA were extracted from murine biopsies followed by assessment of quantity and quality. Results Increased intercellular space between aggregates of myocytes was observed in all samples. This is consistent with reported findings in skeletal muscle using cryogenic conditions. Nonetheless, morphologic analysis of the biopsies proved possible (Figure 2). Optimal recovery of DNA and RNA was obtained from fresh frozen samples as compared to 4% PFA-fixed or 10% formalin-fixed samples. Conclusions This study demonstrates Cryo-X-PCI as a potential tool for analysing myocardial biopsies of heart muscle disease including morphologic analyses. Levels of DNA and RNA were not significantly affected indicating that downstream genomics, proteomics and transcriptomics analyses should be feasible.Setup for Cryo-X-PCI of frozen biopsiesMorphologic analysis of a biopsy

Influence of formalin fixation duration on RNA quality and quantity from formalin-fixed paraffin-embedded hepatocellular carcinoma tissues.

Analyzing RNA samples from formalin-fixed paraffin-embedded (FFPE) tissues is essential for precision medicine. We investigated RNA quantity and quality from FFPE tumor tissues fixed in formalin for various times and compared sequencing metrics from next-generation sequencing (NGS). Hepatocellular carcinoma (HCC) tissues were fixed in 10% neutral buffered formalin (1-240 h) and FFPE blocks were prepared. Total RNA was extracted, and the quantity and quality were assessed using the NanoDrop, Qubit and Bioanalyzer. After preparing sequencing libraries, NGS was performed on the Oncomine Dx Multi-CDx system. Total RNA yields of all samples met the threshold required for NGS, but longer fixation times resulted in decreased total RNA and long RNA fragment (>200 nt) yields. NGS analysis showed fewer sequencing reads of internal control genes from RNA with longer fixation times. RNA extracted from FFPE blocks stored for 500 days had reduced RNA yield and quality compared with RNA obtained from FFPE blocks prepared immediately. In conclusion, short and over-fixation should be avoided because of their negative impact on sequencing quality. Fixation process should be finished promptly within recommended guidelines (6-72 h) for cancer patients.

Comparative assessment of the morphology and antigenicity of human osteochondral units using formalin and coagulant fixatives

Fixation is crucial for preserving tissue integrity during processing, and the most commonly used cross-linking fixative in immunohistochemistry is neutral buffered formalin, which requires antigen retrieval as a crucial step. The successful use of newer coagulant fixatives like methacarn and EMA to preserve isotopes and eliminate the need for antigen retrieval has been reported recently, but their role in decalcified osteochondral unit samples remains unknown. Limited information on the use of coagulant fixatives as formalin substitutes makes it important to comparatively evaluate their effects on osteochondral samples and the impact of antigen retrieval on different days. Osteochondral units from a patient with osteoarthritis who underwent total knee-replacement surgery were fixed with three studied fixatives (Formalin, Methacarn, EMA) and divided into four portions, for different time periods (Day 1, 3, 7, 10). Sections were decalcified, stained with Safranin O, Alcian Blue, Toluidine Blue, PicroSirius Red, Hematoxylin and Eosin, and immunohistochemical analysis of Collagen type II and type X with and without antigen retrieval was conducted. Formalin showed better hematoxylin uptake than coagulant-based fixatives, while all fixatives preserved tissue morphology without necrosis or cellular degeneration with comparable staining quality. Methacarn and EMA-fixed tissues showed higher uptake of collagen type II compared to formalin-fixed tissues, with collagen type II uptake occurring only in the cartilage region and collagen type X uptake occurring only in the bone region. The study highlights the effectiveness of methacarn and EMA as efficient alternatives to formalin, preserving tissue morphology and antigen specificity.

Alternative Tissue Fixation Protocols Dramatically Reduce the Impact of DNA Artifacts, Unraveling the Interpretation of Clinical Comprehensive Genomic Profiling

Formalin-fixed paraffin-embedded (FFPE) samples represent the cornerstone of tissue-based analysis in precision medicine. Targeted next-generation sequencing panels are routinely used to analyze a limited number of genes to guide treatment decision-making for advanced-stage patients. The number and complexity of genetic alterations to be investigated are rapidly growing; in several instances, a comprehensive genomic profiling analysis is needed. The poor quality of genetic material extracted from FFPE samples may impact the feasibility/reliability of sequencing data. We sampled 9 colorectal cancers to allow 4 parallel fixations: (1) neutral buffered formalin (NBF), (2) acid-deprived formalin fixation (ADF), (3) precooled ADF (coldADF), and (4) glyoxal acid free (GAF). DNA extraction, fragmentation analysis, and sequencing by 2 large next-generation sequencing panels (OCAv3 and TSO500) followed. We comprehensively analyzed library and sequencing quality controls and the quality of sequencing results. Libraries from coldADF samples showed significantly longer reads than the others with both panels. ADF-derived and coldADF-derived libraries showed the lowest level of noise and the highest levels of uniformity with the OCAv3 panel, followed by GAF and NBF samples. The data uniformity was confirmed by the TSO500 results, which also highlighted the best performance in terms of the total region sequenced for the ADF and coldADF samples. NBF samples had a significantly smaller region sequenced and displayed a significantly lower number of evaluable microsatellite loci and a significant increase in single-nucleotide variations compared with other protocols. Mutational signature 1 (aging and FFPE artifact related) showed the highest (37%) and lowest (17%) values in the NBF and coldADF samples, respectively. Most of the identified genetic alterations were shared by all samples in each lesion. Five genes showed a different mutational status across samples and/or panels: 4 discordant results involved NBF samples. In conclusion, acid-deprived fixatives (GAF and ADF) guarantee the highest DNA preservation/sequencing performance, thus allowing more complex molecular profiling of tissue samples.

Macroanatomical investigation of sciatic nerve in rat and quail as a model for experimental medical studies: A comparative study of anatomy

Objective: Sciatic nerve studies are still up-to-date from past to present. In recent years, rats and animals other than rats have been used for drugs to be developed to treat the sciatic nerve. This study aims to reveal the comparative anatomy of human, rat and quail and shed light on the experimental studies planned to be done. Material and Methods: 25 rats (Wistar Albino) and Japanese quail (Coturnix japonica) in 10% formalin fixation were dissected for the sciatic nerve. In addition, to show the course of the sciatic nerve in humans, the sciatic nerve was dissected in a male cadaver. Results: In rat, the nerve length between the right and left sides is more significant than the nerve thickness between the right and left sides (p=0.025). A significant difference was found in the correlation graph between the sciatic nerve thickness on the right side and the sciatic nerve thickness on the left side in rats (p=0.006) and quails (p=0.008). Conclusion: Comparative anatomy studies are very valuable for the literature. In particular, the study of a topic such as the sciatic nerve, which is on the agenda, in various experimental animals brings the importance of examining these animals morphometrically. Anatomy of the experimental animals to be studied should also be well known in order to achieve the optimal correct data in the results of correct operation and drug administration.

Ultrasonic properties of fresh and formalin fixed brain tissue

Recent advances in transcranial ultrasound have generated increased interest in the ultrasonic properties of brain. For in vitro laboratory studies of brain tissue, it is convenient to use specimens preserved by formalin fixation. The goal of the present study was to investigate the effect of formalin fixation on the ultrasonic properties of brain. 1-cm thick slices of tissue were prepared from 9 bovine brains. A total of 28 slices from two anatomic planes (16 sagittal, 12 coronal) were studied. Tissue specimens were scanned in phosphate buffered saline at room temperature with a 5 MHz transducer. Scans were performed on fresh tissue and repeated one month after formalin fixation. The speed of sound (SOS) and frequency slope of attenuation (FSA) of ultrasonic pulses propagated through the tissue were determined at every measurement site. The measured values were averaged over all sites on all specimens to obtain the following results expressed as mean ± standard deviation. SOS was (1522 ± 3) m/s before fixation and (1538 ± 12) m/s after fixation. FSA was (0.438 ± 0.064) dB/cm/MHz before fixation and (0.493 ± 0.113) dB/cm/MHz after fixation. Thus, formalin fixation produced approximately a 1% increase in SOS and a 13% increase in FSA.