Formalin Fixation Research Articles

Fluorescence confocal microscopy for margin assessment in prostatectomy: IP8-FLUORESCE study protocol.

Radical prostatectomy (RP) represents the cornerstone of surgical treatment for prostate cancer. Assessing surgical margin status intraoperatively with current techniques remains challenging due to high costs in the context of an already stretched pathology workforce. Fluorescence confocal microscopy (FCM) is a promising technique to detect margins in prostate cancer surgery not bound by such limitations. The Imperial Prostate 8 - Fluorescence Confocal Microscopy for Rapid Evaluation of Surgical Cancer Excision (IP8-FLUORESCE) study is a multicentre, prospective, ex vivo, 'blinded', comparative cohort study. It aims to assess the accuracy of digital FCM for detection of prostate cancer at surgical margins compared to traditional histopathology. The primary endpoint is the accuracy of digital FCM for detection of prostate cancer at surgical margins on a per-patient level, reported with sensitivity, specificity, positive and negative predictive values. A total of 153 patients with localised prostate cancer undergoing robot-assisted RP across three UK National Health Service tertiary referral centres will be recruited. Following RP, prostate specimens will undergo immediate immersion in Acridine Orange solution, scanning 'en face' with FCM using the Histolog® Scanner, and subsequent formalin fixation and paraffin embedding. Two independent, 'blinded' uro-pathologists will report both the FCM images and the histopathology slides. Recruitment commenced on 17 August 2023.

Technical note: Effects of chemical, radiational, and thermal treatment of bone tissue on material stiffness and anisotropy.

We evaluated the effects of four treatments for pathogen inactivation in bone tissue (ethanol storage, formalin fixation, gamma irradiation, and heat treatment via autoclave) on stiffness and anisotropy values in bone samples. Cortical bone samples from the humerus of 14 bovine specimens were subjected to Knoop microindentation analysis in longitudinal and transverse planes of section and four indenter orientations within each section. From each specimen, individual samples were assigned to one of five treatment conditions: 50% ethanol-saline solution, formalin immersion, gamma irradiation, autoclave, and buffered saline (controls). Each sample was microindented 100 times and the resultant stiffness data were analyzed by a resampled factorial analysis of variance. First- and second-order interactions as well as main effects of indenter orientation, treatment, and specimen were significant for both transverse and longitudinal sections, with the sole exception that indenter orientation-treatment interaction was nonsignificant for longitudinal sections. Interaction plots reveal that thermal (autoclave) and irradiation treatments depress stiffness values the most, while patterns of indentation stiffness at different orientations are relatively unaffected. Patterns of anisotropy are relatively unaffected by preservative treatments, but elastic modulus changes are consistent and unambiguous. Formalin and ethanol treatments are most comparable to controls and represent the best preservative media for mechanical testing. These options, however, are likely to be the least effective for ensuring the inactivation or sterilization of potentially contaminated samples.

Factors impacting tumor displacement artifact and pseudo-lymphovascular invasion in testicular germ cell tumors.

Testicular germ cell tumors are susceptible to tumor displacement artifact (TDA), which produces pseudo-lymphovascular invasion (LVI) and confounds the identification of true LVI. Our study aimed to evaluate tumor displacement artifact and pseudo-LVI in testicular germ cell tumors and determine if prolonged fixation improves histological quality. A retrospective search identified 121 orchiectomies with slides that were reviewed to assess TDA and pseudo-LVI. Seminoma had more TDA (68% vs 45%, P= .01) and pseudo-LVI (53% vs 19%, P< .001) than mixed germ cell tumor. Seminoma and mixed germ cell tumor with TDA and pseudo-LVI were larger than those without. Mixed germ cell tumor with ≥50% composition of seminoma had a higher rate of TDA (89% vs 38%, P= .008) and pseudo-LVI (44% vs 15%, P= .06) than those with less. TDA was reduced in seminoma with >1 night fixation compared to no fixation (50% vs 82%, P= .046), with a similar trend in mixed germ cell tumor (31% vs 60%, P= .15). A trend in reduction of pseudo-LVI was seen with >1 night fixation compared to no fixation in seminoma (64% vs 39%, P= .12). Seminomas and larger germ cell tumors were more prone to TDA and pseudo-LVI. Prolonged formalin fixation improved histological quality intesticular germ cell tumors. Based on these data, we recommend fixation for at least 2 nights before sectioning orchiectomy specimens, particularly for larger tumors.

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

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

Anti-Shrinkage Technique for Feline Skin Samples

Surgical resection of subcutaneous neoplasms with clear margins is crucial for preventing local recurrence and avoiding adjuvant treatments. However, the evaluation of surgical margins often differs significantly from the histopathological assessment due to tissue shrinkage, which can result in inaccurate therapeutic assessments and unreliable patient prognoses. In this study, ten feline cadavers were utilized. Six 50 mm diameter specimens were collected from three regions (thorax, flank, femur) and measured at three time points: T0 (excision time), T1 (10 min after incision), and T2 (at least 48 h after sample collection and formalin fixation). Samples in the study group were stretched and fixed on a cork plate with pinpoint needles after excision to restore their original dimensions. All specimens exhibited a similar trend. After 48 h of formalin fixation, the control specimens showed significant shrinkage, with a reduction of 25.73% in radius and 26.32% in diameter. In contrast, the study specimens demonstrated minimal changes, with a radius reduction of −0.28% and no change in diameter. The results indicate that all feline skin specimens experienced significant shrinkage of approximately one-quarter from their pre-incisional size. Stretching and pinning the excised tissues allowed for the restoration and maintenance of original dimensions even after formalin fixation. This technique represents a valid and practical approach to minimize tissue shrinkage.

Accuracy of the LaserSAFE technique for detecting positive surgical margins during robot-assisted radical prostatectomy: blind assessment and inter-rater agreement analysis.

Fluorescence confocal microscopy (FCM) is a new imaging modality capable of generating digital microscopic resolution scans of fresh surgical specimens, and holds potential as an alternative to frozen section (FS) analysis for intra-operative assessment of surgical margins. Previously, we described the LaserSAFE technique as an application of FCM for margin assessment in robot-assisted radical prostatectomy (RARP) using the Histolog® scanner. This study describes the accuracy and inter-rater agreement of FCM imaging compared to corresponding paraffin-embedded analysis (PA) among four blinded pathologists for the presence of positive surgical margins (PSM). RARP specimens from patients enrolled in the control arm of the NeuroSAFE PROOF study (NCT03317990) were analysed from April 2022 to February 2023. Prostate specimens were imaged using the Histolog® scanner before formalin fixation and PA. Four trained assessors, blinded to PA, reviewed and analysed FCM images of the posterolateral prostatic surface. A total of 31 prostate specimens were included in the study. PA per lateral side of the prostate identified 11 instances of positive margins. Among the four histopathologists included in our study, FCM achieved a sensitivity of 73-91 and specificity of 94-100% for the presence of PSM. Fleiss' Kappa for inter-rater agreement on PSM was 0.78 (95% confidence interval = 0.64-0.92), indicating substantial agreement. This blinded analysis of FCM versus PA among histopathologists with different experience levels demonstrated high accuracy and substantial inter-rater agreement for diagnosing PSM. This supports the role of the FCM as an alternative to FS.

Forensic STR and SNP Genotyping of Formalin-Fixed Skeleton Samples With Illumina's ForenSeq System.

Formalin fixatives are widely used in forensics to preserve tissues. However, extracting high-quality genomic DNA from formalin-fixed samples is challenging. Traditional short tandem repeat (STR) analysis using capillary electrophoresis (CE) for forensic DNA typing frequently results in failure. Massively parallel sequencing (MPS) can handle many samples and thousands of genetic markers, usually single-nucleotide polymorphisms (SNPs) and STRs, in a single test. Thus, it is useful for assessing highly deteriorated forensic evidence. Few studies have examined the effectiveness of STRs and SNPs genotyping of formalin-fixed skeletons using MPS. In this study, 55 skeletal samples from 5 individuals that were treated under different formalin fixation times (5-75 days) were examined and sequenced using the ForenSeq DNA Signature Prep Kit on the Illumina MiSeq FGX platform. The results showed that as the duration of formalin fixation increased, the detection rates of STRs and SNPs gradually decreased. After 75 days of fixation, the average detection rates for STRs and SNPs were 4% and 10%, respectively. The cumulative discrimination power (CDP) of individual identification SNPs (iiSNPs) was >0.9999 on the 45th day. However, the CDP of STRs was 0.9930 on the 22nd day. Low detection rates were observed for six STRs (D1S1656, PentaE, D22S1045, PentaD, DX8378 and DX10103) and five SNPs (rs2920816, rs354439, rs1736442, rs338882 and rs1031825). In conclusion, DNA extracted from formalin-fixed skeletons decomposes rapidly over time, and MPS technology can be a useful tool for detecting forensic genetic markers in such samples.

Method for co-registration of high-resolution specimen PET-CT with histopathology to improve insight into radiotracer distributions

BackgroundAs the spatial resolution of positron emission tomography (PET) scanners improves, understanding of radiotracer distributions in tissues at high resolutions is important. Hence, we propose a method for co-registration of high-resolution ex vivo specimen PET images, combined with computed tomography (CT) images, and the corresponding specimen histopathology.MethodsWe applied our co-registration method to breast cancer (BCa) specimens of patients who were preoperatively injected with 0.8 MBq/kg [18\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$^{18}$$\\end{document}F]fluorodeoxyglucose ([18F]FDG). The method has two components. First, we used an image acquisition scheme that minimises and tracks tissue deformation: (1) We acquired sub-millimetre (micro)-PET-CT images of ±2 mm-thick lamellas of the fresh specimens, enclosed in tissue cassettes. (2) We acquired micro-CT images of the same lamellas after formalin fixation to visualise tissue deformation. (3) We obtained 1 hematoxylin and eosin (H&E) stained histopathology section per lamella of which we captured a digital whole slide image (WSI). Second, we developed an automatic co-registration algorithm to improve the alignment between the micro-PET-CT images and WSIs, guided by the micro-CT of the fixated lamellas. To estimate the spatial co-registration error, we calculated the distance between corresponding microcalcifications in the micro-CTs and WSIs. The co-registered images allowed to study standardised uptake values (SUVs) of different breast tissues, as identified on the WSIs by a pathologist.ResultsWe imaged 22 BCa specimens, 13 cases of invasive carcinoma of no special type (NST), 6 of invasive lobular carcinoma (ILC), and 3 of ductal carcinoma in situ (DCIS). While the cassette framework minimised tissue deformation, the best alignment between the micro-PET-CT images and WSIs was achieved after deformable co-registration. We found an overall average co-registration error of 0.74 ± 0.17 mm between the micro-PET images and WSIs. (Pre)malignant tissue (including NST, ILC, and DCIS) generally showed higher SUVs than healthy tissue (including healthy glandular, connective, and adipose tissue). As expected, inflamed tissue and skin also showed high uptake.ConclusionsWe developed a method to co-register micro-PET-CT images of surgical specimens and WSIs with an accuracy comparable to the spatial resolution of the micro-PET images. While currently, we only applied this method to BCa specimens, we believe this method is applicable to a wide range of specimens and radiotracers, providing insight into distributions of (new) radiotracers in human malignancies at a sub-millimetre resolution.

Outcomes of Neoadjuvant Chemotherapy in Locally Advanced Oral Cancers: A Retrospective Analytical Study.

Introduction Head and neck cancers account for 30% of malignancies in India and oral squamous cell carcinoma is most common. 80% of the patients present with locally advanced disease and many of them are inoperable. In this study we documented the outcomes of neoadjuvant chemotherapy (NACT) in locally advanced oral cancer staged T4 with regards to downstaging the disease and to make the tumor more amenable for surgery with better access. The objectives of the study are to document the outcomes of NACT in patients with locally advanced oral cancers and to correlate the response to NACT and adequacy of resection by histopathology examination of the specimen. Methodology Clinical data of 96 patients with locally advanced squamous cell carcinoma of the oral cavity staged T4a and T4bwho received two cycles of NACT between December 2018 and November 2022 were reviewed respectively and the outcomes and response following NACT were documented. Results Among 96 patients in this study, 68 (70.9%) patients were staged T4a and 28 (29.1%) were staged T4b. 31 (30.2%) patients showed partial response, 36 (37.5%) had stable disease and 29 (30.2%) had progressive disease following 2 cycles of NACT. All patients underwent definitive surgery following NACT and were followed up for 14 months. Among those staged T4a, 12 patients (33.3%)had close margins, eight patients (34.8%) had positive margins and 10 patients (16.2%) had a recurrence. Among those staged T4b, 24 patients (66.7%) had close margins (less than 5mm distance after formalin fixation), 15 patients (65.2%) had positive margins while 17 patients (60.7%) had a recurrence in the 14 month follow-up period. Conclusion NACT makes tumors more amenable for surgery in locally advanced oral squamous carcinoma. However, 30% of patients do not respond to it.

Atrial septal pouch in cats: first report on interatrial septum anatomical variations.

The atrial septal pouch was first recognised in humans in 2006 and, since 2010, has been described in more detail. The first reports on the left-sided atrial septal pouch linked its occurrence with a higher risk of thromboembolic stroke resulting from left atrial thrombus formation. However, subsequent studies have challenged this theory; therefore, the consequences of septal pouch occurrence remain disputed. Currently, in veterinary species, septal pouch occurrence and morphology have been described in sheep and pigs, showing species-related differences. No studies have been conducted on cats. As cats show a species-related risk of aortic thromboembolic disease resulting from left atrial thrombus formation, we aimed to describe the interatrial septum anatomical variations as the first step in the investigation of the relationship between the septal pouch presence and left atrial thrombus formation. The study was conducted on hearts collected post mortem from 80 cats. After formalin fixation and dissection, a detailed anatomical description of the interatrial septum for all cases was followed by a histopathological analysis of the heart in 25 cases. After dissection, the specimens were embedded in paraffin blocks and stained with haematoxylin and eosin, Masson-Goldner trichrome, Picrosirius Red and Movat pentachrome in a routine manner and evaluated via light microscopy. Our study showed a very high prevalence (95%) of the left-sided septal pouch in cats with a lower incidence (29%) of the right-sided septal pouch. We noted a high morphological variety of the left-sided septal pouch and described anatomical variants not previously reported in the literature. The septal pouch is a common anatomical variation of the interatrial septum in cats. Although, based on the current study, we cannot draw further conclusions on the relationship between the left-sided septal pouch and thromboembolic events in cats, the high prevalence of this anatomical entity points to a need of further studies on the topic.

Cutaneous tissue samples show significant shrinkage during histopathological work-up: a real-world study.

Excision and histological examination of cutaneous neoplasms are very common diagnostic and therapeutic procedures in dermatological practice. There are often discrepancies between tissue seize in vivo and after histopathological work-up. This may raise questions according to tumor sizes or safety margins. To investigate the effect of excision and histological processing on the properties of cutaneous neoplasms in real-life 401 specimens in 324 patients were prospectively investigated. Delineated excision margins of cutaneous neoplasms were documented prior to (T0) and post excision (T1). Length, width and thickness were measured after ≥48 h of formalin fixation (T2) and after sectioning (T3) on final histological slices. Accompanying parameters such as patient age, gender, anatomical site, and tumor entity were evaluated. All post-processed tissue exhibited a significant (P<0.001) median (interquartile range) reduction in length, width and thickness of 21.0% (9.5-30.0), 30.7% (20.0-40.0), and 28.0% (4.8-46.7), respectively, irrespective of site, patient age or tumor entity. Maximum median (IQR) reduction for the length and the width was observed right after excision (17.0% [4.3-25.0] and 14.0% [11.1-28.6] reduction). No significant median (IQR) tissue changes between T1 and T2 were observed (length: 4.8% (-4.3-13.3); width: 0% (-17.6-11.1); thickness 0% (-32.0-20.0). Subgroup analyses showed significantly greater tissue shrinkage in younger patients and for tissue sample sites (trunk or lower extremities). Most relevant shrinkage of cutaneous samples occurs right after excision. Age- and site-depended tissue contractility can influence these effects. Formalin fixation does not affect tissue shrinkage. Smaller tissue sizes on histopathological reports are to be expected.

Acute effect of the novel cadaver fixation and storage solution -Anatomical-biomechanical-microbiological study

Cadaver education is a golden standard in health sciences. It is aimed to purify the cadavers from microbes by subjecting them to chemicals and to be used for many years by terminating autolysis. As a result of literature searches, a solution consisting of a mixture of borax, nitrite-nitrate, glycerin, alcohol, and thyme oil was created. While seven sheep hearts were placed in this solution, seven were kept in 10% formaldehyde for two months for fixation and storage. At the end of the first ten days, it was observed that the discoloration of the heart in the solution was lower than the formalin fixation. In histological examinations, it was observed that there was no visual difference between the groups and the tissue could be examined at all magnifications. At the end of two months, insignificant microbial growth observed in the solution. While color changes were better than formaldehyde, texture profile analysis values were found to be closer to fresh heart. In histological examinations, in the solution group, although the tissue could be recognized at all magnifications, it was observed that cell nuclei were less stained at x100. It was concluded that this solution can be used mainly in short-term courses.

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

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

A study of PGP 9.5 immunohistochemical labeling on formalin-fixed paraffin embedded tissues for epidermal nerve fiber density testing

ABSTRACT There have been several methods established for immunohistochemical labeling of the PGP 9.5 antigen in human tissue for the assessment of epidermal nerve fiber density, none of which uses neutral-buffered formalin as the preferred fixative for paraffin-embedded tissue. The literature maintains that formalin-fixed paraffin embedded tissues are unable to be used for this purpose and that other fixatives must be used due to the cross-linkages caused by formalin fixation. This study was undertaken to develop a standardized method for the use of formalin-fixed paraffin embedded tissues for immunohistochemical labeling and assessment of epidermal nerve fiber density. Formalin-fixed paraffin embedded tissues from the punch biopsies of patients suspected to have small fiber neuropathy were prepared for immunohistochemical labeling using heat-induced epitope retrieval for one hour at 92°C. The tissues were then stained with a polyclonal rabbit anti-human PGP 9.5 primary antibody. The resulting stains were then evaluated by a licensed pathologist who counted the number of epidermal nerve fibers stained and recorded the epidermal length in millimeters. Human foreskin was used as the tissue control in these studies. Satisfactory immunohistochemical labeling of epidermal nerve fibers was achieved from formalin-fixed paraffin embedded tissues through the use of heat-induced epitope retrieval. The authors of this paper have concluded that formalin-fixed paraffin embedded tissues may be used to achieve satisfactory immunohistochemical labeling for the assessment of epidermal nerve fiber density.

Comparative analysis of in situ and ex situ postmortem brain MRI: Evaluating volumetry, DTI, and relaxometry

AbstractPurposeTo compare postmortem in situ with ex situ MRI parameters, including volumetry, diffusion tensor imaging (DTI), and relaxometry for assessing methodology‐induced alterations, which is a crucial prerequisite when performing MRI biomarker validation.MethodsMRI whole‐brain scans of five deceased patients with amyotrophic lateral sclerosis were performed at 3 T. In situ scans were conducted within 32 h after death (SD 18 h), and ex situ scans after brain extraction and 3 months of formalin fixation. The imaging protocol included MP2RAGE, DTI, and multi‐contrast spin‐echo and multi‐echo gradient‐echo sequences. Volumetry, fractional anisotropy, mean diffusivity, T1, T2, and have been assessed for specific brain regions.ResultsWhen comparing ex situ to in situ values, the following results were obtained. Deep gray matter as well as the thalamus and the hippocampus showed a reduced volume. Fractional anisotropy was reduced in the cortex and the whole brain. Mean diffusivity was decreased in white matter and deep gray matter. T1 and T2 were reduced in all investigated structures, whereas was increased in the cortex.ConclusionThe results of this study show that the volumes and MRI parameters of several brain regions are potentially affected by tissue extraction and subsequent formalin fixation, suggesting that methodological alterations are present in ex situ MRI. To avoid overlap of indistinguishable methodological and disease‐related changes, we recommend performing in situ postmortem MRI as an additional intermediate step for in vivo MRI biomarker validation.

Retrospective Analyses of Porcine Circovirus Type 3 (PCV-3) in Switzerland.

Porcine circovirus 3 (PCV-3) has emerged as a significant pathogen affecting global swine populations, yet its epidemiology and clinical implications remain incompletely understood. This retrospective study aimed to investigate the prevalence and histopathological features of PCV-3 infection in pigs from Switzerland, focusing on archival cases of suckling and weaner piglets presenting with suggestive lesions. An in-house qPCR assay was developed for detecting PCV-3 in frozen and formalin-fixed paraffin-embedded tissues, enhancing the national diagnostic capabilities. Histopathological reassessment identified PCV-3 systemic disease (PCV-3-SD) compatible lesions in 19 (6%) of archival cases, with 47% testing positive by qPCR across various organs. Notably, vascular lesions predominated, particularly in mesenteric arteries, heart, and kidneys. The study confirms the presence of PCV-3 in Switzerland since at least 2020, marking the first documented cases within the Swiss swine population. Despite challenges in in situ hybridization validation due to prolonged formalin fixation, the findings indicate viral systemic dissemination. These results contribute to the understanding of PCV-3 epidemiology in Swiss pigs, emphasizing the need for continued surveillance and further research on its clinical implications and interaction with host factors. Our study underscores the utility and limitations of molecular techniques in confirming PCV-3 infections.

Large field of view and spatial region of interest transcriptomics in fixed tissue

Expression profiling in spatially defined regions is crucial for systematically understanding tissue complexity. Here, we report a method of photo-irradiation for in-situ barcoding hybridization and ligation sequencing, named PBHL-seq, which allows targeted expression profiling from the photo-irradiated region of interest in intact fresh frozen and formalin fixation and paraffin embedding (FFPE) tissue samples. PBHL-seq uses photo-caged oligodeoxynucleotides for in situ reverse transcription followed by spatially targeted barcoding of cDNAs to create spatially indexed transcriptomes of photo-illuminated regions. We recover thousands of differentially enriched transcripts from different regions by applying PBHL-seq to OCT-embedded tissue (E14.5 mouse embryo and mouse brain) and FFPE mouse embryo (E15.5). We also apply PBHL-seq to the subcellular microstructures (cytoplasm and nucleus, respectively) and detect thousands of differential expression genes. Thus, PBHL-seq provides an accessible workflow for expression profiles from the region of interest in frozen and FFPE tissue at subcellular resolution with areas expandable to centimeter scale, while preserving the sample intact for downstream analysis to promote the development of transcriptomics.

The influence of fixing condition on myoglobin stainability of striated muscle as a tool for forensic diagnosis

It is reported that immunostaining of Myoglobin (Mb) is useful for forensic diagnosis. In this study, we investigated the condition of fixation of striated muscle in 10 % neutral-buffered formalin to obtain appropriate stationarity of Mb in immunostaining. Firstly, criteria for staining intensity and definition of the stainability of examined were determined for sheep muscle tissue. Sheep myocardial tissue was fixed using 10 % neutral-buffered formalin under the 21 different conditions based on combinations of the following: three ratios of volume of formalin (mL) to weight of myocardium (g) (RFM) of 1, 4 or 9, 7 durations of fixation (DF) of 0.5, 3 or 6 h, and 1, 2, 5 or 7 days. Secondly, detection of Mb diffused form skeletal muscle from autopsy cases into formalin during fixation were confirmed by ELISA. Finally, the evaluation of stainability of Mb of striated muscle in routine autopsy examinations was confirmed using sheep staining intensity standards. From this experimental investigation, it has been demonstrated that the most suitable formalin fixation condition for using Mb staining in forensic diagnosis is RFM4 with a fixation time of at least DF 3 h up to 1 day. It was evident that staining intensity decreases with fixation durations exceeding 2 days, irrespective of the RFM. Thus, the fixation time was deemed the most influential factor affecting the staining properties of Mb staining in skeletal muscle tissue. When conducting Mb staining using striated muscle as an evaluation sample, particular attention should be paid to the fixation time.

The particularities of connective fibers from the wall of varicose veins extirpated by cryostripping.

The varicose vein affects more than 30% of the general population. Significantly increased rates were noticed in women and older population. From the histopathological point of view, venous arterialization, smooth muscle cell hypertrophy, and hyperplasia are the main changes noticed in varicose vein disease. Some of the main therapeutic methods used in the management of varicose disease are injection sclerotherapy, conservative, surgical, saphenous vein inversion and removal, high saphenous ligation, ambulatory phlebectomy, transilluminated powered phlebectomy, endovascular management, cryostripping. The aim of this study was to evaluate the morphology of connective fibers from the wall of the varicose veins extirpated by cryostripping. The study included 109 samples taken by cryostripping method. Hematoxylin-Eosin, Masson's trichrome, Silver and Orcein staining were applied. The assessment of fibers was made according to score values between 0 and 3. It was found no major structural differences in terms of alterations of collagen fibers induced by the applied surgical procedure. It was noticed duplications and multiplications of the internal elastic lamina, as diffuse and nodular forms. Depletion of elastic fibers at the media was a lesion identified in most of the specimens. The depletion of reticulin fibers correlates with the accumulation of collagen fibers, which partially or completely replace the network in the media and intima. No correlation was found between changes in the reticulin network and the time between prelevation and buffered formalin fixation, the maximum time investigated being five days. The Orcein staining in the venous vessel evaluation panel may be a useful investigation.

Impairment of the GABAergic system in the anterior insular cortex of heroin-addicted males.

Opioid addiction is a global problem, causing the greatest health burden among drug use disorders, with opioid overdose deaths topping the statistics of fatal overdoses. The multifunctional anterior insular cortex (AIC) is involved in inhibitory control, which is severely impaired in opioid addiction. GABAergic interneurons shape the output of the AIC, where abnormalities have been reported in individuals addicted to opioids. In these neurons, glutamate decarboxylase (GAD) with its isoforms GAD 65 and 67 is a key enzyme in the synthesis of GABA, and research data point to a dysregulation of GABAergic activity in the AIC in opioid addiction. Our study, which was performed on paraffin-embedded brains from the Magdeburg Brain Bank, aimed to investigate abnormalities in the GABAergic function of the AIC in opioid addiction by densitometric evaluation of GAD 65/67-immunostained neuropil. The study showed bilaterally increased neuropil density in layers III and V in 13 male heroin-addicted males compared to 12 healthy controls, with significant U-test P values for layer V bilaterally. Analysis of confounding variables showed that age, brain volume and duration of formalin fixation did not confound the results. Our findings suggest a dysregulation of GABAergic activity in the AIC in opioid addiction, which is consistent with experimental data from animal models and human neuroimaging studies.