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Autoimmune pancreatitis (AIP) is a disease concept that originated in Japan. It is characterized by diffuse pancreatic enlargement and irregular narrowing of the main pancreatic duct. Although the usefulness of the histological diagnosis of AIP using endoscopic ultrasound (EUS)-guided fine-needle aspiration (FNA) and EUS-guided fine-needle biopsy (FNB) has been reported, enhanced diagnostic performance is expected with improvements in tissue collection methods and fine-needle techniques. Guidance for establishing the tissue diagnosis of AIP has been developed and is useful for histological evaluation. Histopathological diagnosis by EUS-FNA/FNB is expected to play a central role in AIP diagnosis in the future.

Clinical cancer genomic testing based on next-generation sequencing can help select genotype-matched therapy and provide diagnostic and prognostic information. Pathological tissue from malignant tumors obtained during routine practice are frequently used for genomic testing. This article is aimed to standardize the proper handling of pathological specimens in practice for genomic medicine based on the findings established in "Guidelines on the handling of pathological tissue samples for genomic medicine (in Japanese)" published by The Japanese Society of Pathology (JSP) in 2018. The two-part practical guidelines are based on empirical data analyses; Part 1 describes the standard preanalytic operating procedures for tissue collection, processing, and storage of formalin-fixed paraffin-embedded (FFPE) samples, while Part 2 describes the assessment and selection of FFPE samples appropriate for genomic testing, typically conducted by a pathologist. The guidelines recommend that FFPE sample blocks be used within 3 years from preparation, and the tumor content should be >= 30% (minimum 20%). The empirical data were obtained from clinical studies performed by the JSP in collaboration with leading Japanese cancer genome research projects. The Japanese Ministry of Health, Labour, and Welfare (MHLW) recommended to comply with the JSP practical guidelines in implementing cancer genomic testing under the national health insurance system in over 200 MHLW-designated core and cooperative cancer genome medicine hospitals in Japan.

Background Following surgical and adjuvant treatment of primary colorectal cancer, many patients are routinely followed up with axial imaging (most commonly computerised tomography imaging) and blood carcinoembryonic antigen (a tumour marker) testing. Because fewer than one-fifth of patients will relapse, a large number of patients are followed up unnecessarily. Objectives To determine whether or not the intratumoural immune signature could identify a cohort of patients with a relapse rate so low that follow-up is unnecessary. Design An observational study based on a secondary tissue collection of the tumours from participants in the FACS (Follow-up After Colorectal Cancer Surgery) trial. Setting and participants Formalin-fixed paraffin-embedded tumour tissue was obtained from 550 out of 1202 participants in the FACS trial. Tissue microarrays were constructed and stained for cluster of differentiation (CD)3+ and CD45RO+ T lymphocytes as well as standard haematoxylin and eosin staining, with a view to manual and, subsequently, automated cell counting. Results The tissue microarrays were satisfactorily stained for the two immune markers. Manual cell counting proved possible on the arrays, but manually counting the number of cores for the entire study was found to not be feasible; therefore, an attempt was made to use automatic cell counting. Although it is clear that this approach is workable, there were both hardware and software problems; therefore, reliable data could not be obtained within the time frame of the study. Limitations The main limitations were the inability to use machine counting because of problems with both hardware and software, and the loss of critical scientific staff. Findings from this research indicate that this approach will be able to count intratumoural immune cells in the long term, but whether or not the original aim of the project proved possible is not known. Conclusions The project was not successful in its aim because of the failure to achieve a reliable counting system. Future work Further work is needed to perfect immune cell machine counting and then complete the objectives of this study that are still relevant. Trial registration Current Controlled Trials ISRCTN41458548. Funding This project was funded by the National Institute for Health Research (NIHR) Health Technology Assessment programme and will be published in full in Health Technology Assessment; Vol. 25, No. 2. See the NIHR Journals Library website for further project information.

Gene expression studies are reported to be influenced by pre-analytical factors that can compromise RNA yield and integrity, which in turn may confound the experimental findings. Here we investigate the impact of four pre-analytical factors on brain-derived RNA: time-before-collection, tissue specimen size, tissue collection method, and RNA isolation method. We report no significant differences in RNA yield or integrity between 20 mg and 60 mg tissue samples collected in either liquid nitrogen or the RNAlater stabilizing solution. Isolation of RNA employing the TRIzol reagent resulted in a higher yield compared to isolation via the QIAcube kit while the latter resulted in RNA of slightly better integrity. Keeping brain tissue samples at room temperature for up to 160 min prior to collection and isolation of RNA resulted in no significant difference in yield or integrity. Our findings have significant practical and financial consequences for clinical genomic departments and other laboratory settings performing large-scale routine RNA expression analysis of brain samples.

Background Glioblastomas (GBMs) are known for having a vastly heterogenous histopathology. Several studies have shown that GBMs can be histologically undergraded due to sampling errors of small tissue samples. We sought to explore to what extent histological features in GBMs are dependent on the amount of viable tissue on routine slides from both biopsied and resected tumors. Methods In 106 newly diagnosed GBM patients, we investigated associations between the presence or degree of 24 histopathological and two immunohistochemical features and the tissue amount on hematoxylin-eosin (HE) slides. The amount of viable tissue was semiquantitatively categorized as “sparse,” “medium,” or “substantial” for each case. Tissue amount was also assessed for associations with MRI volumetrics and the type of surgical procedure. Results About half (46%) of the assessed histological and immunohistochemical features were significantly associated with tissue amount. The significant features were less present or of a lesser degree when the tissue amount was smaller. Among the significant features were most of the features relevant for diffuse astrocytic tumor grading, i.e., small necroses, palisades, microvascular proliferation, atypia, mitotic count, and Ki-67/MIB-1 proliferative index (PI). Conclusion A substantial proportion of the assessed histological features were at risk of being underrepresented when the amount of viable tissue on HE slides was limited. Most of the grading features were dependent on tissue amount, which underlines the importance of considering sampling errors in diffuse astrocytic tumor grading. Our findings also highlight the importance of adequate tissue collection to increase the quality of diagnostics and histological research.

Tissue Biobanks represent an invaluable resource. Despite the majority of people supporting tissue donation, the actual rate remains low overall. Tissue discarded from surgical procedures represents a further avenue for collection for use in research. We aim to understand the information and consent requirements in a cohort of healthy, post-ophthalmic surgical subjects to optimise future tissue collection in living donors. Patients attending an ophthalmic clinic following refractive surgery for myopia (SMILE) were identified. Patient consent was implied with the completion of the provided survey. The questionnaire included gender, age range and education status. The majority of 31 subjects identified a benefit for future patients as the main motive for potential donation of discarded tissue (71%). Payment for the discarded tissue would not influence their decision in 77.4%. Explanation of the potential benefits of research was the most important information to consider before making a decision to donate. Only 12.9% of patients would have refused to include further information. Almost half of patients felt that the Biobank became the owner of tissue following donation. Current surgical patients may be more inclined to participate in research than the general public because of a sense of duty or an increased understanding of the role of research in evolving treatment. Despite minor uncertainty about the eventual use of the tissue and data, most subjects were positive to donation of discarded ocular tissue and de-identified information. Consent and education processes should be revised within an ophthalmic practice to minimise future patient anxiety.

The microbiota isolated from the urine of bladder carcinoma patients exhibits significantly increased compositional abundance of some bacterial genera compared to the urine of healthy patients. Our aim was to compare the microbiota composition of cancerous tissues and urine samples collected from the same set of patients in order to improve the accuracy of diagnostic measures. Tissue samples were collected from patients during cancer tissue removal by transurethral resection. In parallel, urine samples were obtained by transurethral resectoscopy from the same patients. The V3–V4 region of the bacterial 16S rRNA gene was sequenced and analyzed using the Kraken pipeline. In the case of four patients, duplicate microbiota analysis from distant parts of the cancerous tissues was highly reproducible, and independent of the site of tissue collection of any given patient. Akkermansia, Bacteroides, Clostridium sensu stricto, Enterobacter and Klebsiella, as “five suspect genera”, were over-represented in tissue samples compared to the urine. To our knowledge, this is the first study comparing urinary and bladder mucosa-associated microbiota profiles in bladder cancer patients. More accurate characterization of changes in microbiota composition during bladder cancer progression could provide new opportunities in the development of appropriate screening or monitoring methods.

Many biological processes are partitioned among organs and tissues, necessitating tissue-specific or organ-specific analysis (particularly for comparative -omics studies). Standardised techniques for tissue identification and dissection are therefore imperative for comparing among studies. Here we describe dissection protocols for isolating six key tissues/organs from larvae of the Asian longhorned beetle, Anoplophora glabripennis (Motschulsky) (Coleoptera: Cerambycidae): the supraoesophageal ganglion, posterior midgut, hindgut, Malpighian tubules, fat body, and thoracic muscle. We also describe how to extract haemolymph and preserve whole larvae for measurements such as protein, lipid, and carbohydrate content. We include dissection protocols for both fresh-killed and previously frozen specimens. Although this protocol is developed for A. glabripennis, it should allow standardised tissue collection from larvae of other cerambycids and be readily transferrable to other beetle taxa with similar larval morphology.

Background There have been a few cases of echogenic cardiac implantable electric device (CIED) lead‐associated oscillating intracardiac masses (ICMs) in leads imaged by echocardiography. The histological properties of ICMs could help clarify the etiological diagnosis. Although there is extensive literature on mass size, the histological properties of such masses have not been characterized. The aim of this research was to clarify the histological features of oscillating ICMs in CIED patients. Methods Preoperative echocardiography was performed in all candidates for CIED removal. In the patients with ICMs, specimens were obtained by 3 methods: direct tissue collection during open‐heart surgery; tissue collection together with the CIED lead during transvenous extraction; and tissue collection by catheter vacuum during transvenous CIED removal. A standard histopathological examination of ICM tissue was performed. Results A total of 106 patients underwent lead removal in our institute (April 2009‐March 2018); 14 patients had an ICM (13.2%), and 7 specimens were obtained in patients with CIED lead‐related ICM. Following histological examination, 2 types of ICM were identified: one mainly composed of thickened endocardium (EN type; 3 patients), and the other mainly an aggregate of inflammatory cells as a neutrophil cell (NC type; 4 patients). Conclusions Two histological types of intracardiac masses, including a thickened endocardium type and a neutrophil cell type, were identified. These classifications might help make an accurate histological diagnosis of lead‐associated intracardiac masses., This research was to clarify the histological features of oscillating ICMs in CIED patients. Two histological types of intracardiac masses, including a thickened endocardium type and a neutrophil cell type, were identified. These classifications might help make an accurate histological diagnosis of lead‐associated intracardiac masses.

Newer ‘omics approaches such as metatranscriptomics and metabolomics allow functional assessments of the interaction(s) between the gut microbiome and the human host. In order to generate meaningful data with these approaches, though, the method of sample collection is critical. Prior studies have relied upon expensive and invasive means towards sample acquisition such as intestinal biopsy, while other studies have relied upon easier methods of collection such as fecal samples that do not necessarily represent those microbes in contact with the host. In this pilot study, we attempt to characterize a novel, minimally invasive method towards sampling the human microbiome using mucosal cytology brush sampling compared to intestinal gut biopsy on 5 healthy participants undergoing routine screening colonoscopy. We compared metatranscriptomic analyses between the two collection methods, identifying increased taxonomic evenness and beta diversity in the cytology brush samples, and similar community transcriptional profiles between the two methods. Metabolomics assessment demonstrated striking differences between the two methods, implying a difference in bacterial-derived versus human absorbed metabolites. Put together, this study supports the use of a less invasive method of microbiome sampling with cytology brushes, but caution must be exercised when performing metabolomics assessment as this represents differential metabolite production but not absorption by the host.ImportanceIn order to generate meaningful metabolomic and microbiome data, the method of sample collection is critical. This study utilizes and compares two methods to intestinal tissue collection for evaluation of metabolites and microbiome, finding that using a brush to sample the microbiome is superior to tissue biopsy. However, for metabolomics assessment, biopsy may still be required.

Precision tissue diagnostics rely on high quality input specimens so that assay results are not affected by artifact, but advances in collection and processing of tissue specimens have lagged behind innovations in diagnostic assay development. Therefore, we have designed and evaluated a novel surgical tissue collection device that maintains and monitors sample temperature and motion throughout transport so that the major preanalytical variable of tissue temperature can be controlled and measured. This device, in combination with an improved cold–hot tissue fixation protocol affords optimal biomarker preservation in less overall time, thereby simultaneously improving diagnostic quality and turnaround time. We collected 50 primary and metastatic liver tumors using a novel transport device. Tissue was fixed using a rapid cold–hot fixation protocol and immunohistochemical assays were used to assess the performance of the device, in comparison to control tissue preserved using standard clinical fixation protocol. Two pathologists evaluated the IHC studies in a blinded fashion to determine the immunophenotype of each tumor. The observed IHC staining intensities and the clinical impressions of the immunophenotypes did not differ between tissue collected with the novel device and control tissue, while improvements in processing time were achieved. The novel cold transport device and rapid fixation protocol can be successfully and safely combined and used to monitor specimen conditions, thus preserving the diagnostic utility of specimens and improving the overall turn-around time of the diagnostic process. Electronic supplementary material The online version of this article (10.1007/s10561-019-09800-8) contains supplementary material, which is available to authorized users.

Bats are becoming increasingly important as an experimental model due to their unique biological features. These include the ability of powered flight, minimal consequences from a heightened metabolic state, extended longevity in most species and minimal inflammation in response to most otherwise pathogenic viruses. To date there has been limited work done on the optimal procedures for necropsy, extraction of tissues or preparation of cell suspensions for downstream experimental work. Here we use Pteropus alecto black flying fox as an example model of the fruit bat to develop optimal procedures for anaesthetizing, necropsy methods, safety, sequence and protocols for cell/tissue extraction and isolation protocols. These methods were successfully used to yield high-quality RNA, DNA and protein samples from tissues along with viable cells for various molecular and immunological studies. Procedures utilized are suitable for comparative biology studies with most protocols being directly modified from those used in mice and humans. While mainly optimized for the larger fruit bats (flying foxes) in this study, the majority of protocols can readily be adapted to all species of bats. This study provides the framework for greater consistency with in vivo bat experiments, application for comparative biology studies and greater engagement of the bat community for suitable protocols to be harmoniously adopted.

While the state of the art has been described for recording bird songs and calls, there are no described best practices for collecting and preserving the avian vocal organ, the syrinx. In addition to skins and skeletal preparations, field collection of tissues for DNA sequencing has become a common practice. Protocols for such tissue collection often involve accessing internal organs. Here we argue that collecting syringes, which also involves accessing the chest cavity, adds only minimal time to field or skeletal preparation and can be done without damage to the skeleton. Collecting syringes will enable studies of structure–function relationships in the production of calls and song that have not been previously possible. Three-dimensional X-ray computed tomography (CT) imaging techniques allow new insight into these relationships, but material of both male and female birds as well as juveniles is lacking for most avian taxa. Here, we present a guide to the removal and preservation of the avian syrinx and associated vocal anatomy in hopes of increasing their representation in biological collections. In this guide, we suggest that taking in situ measurements is ideal since dissection, fixation, and tissue staining can affect measurements of certain airway features such as tracheal diameter and bronchial length. Our guidelines demonstrate that removing a single wing, shoulder, and half of the pectoral girdle from the dorsal side of the thorax along with disarticulating the sternal and vertebral rib segments on one side of the body allows exposure of the complete airway without damaging the skeleton. We also provide recommendations for fixation, storage, and staining of specimens in preparation for diffusible iodine-based contrast-enhanced computed tomography (diceCT) scans that allow imaging of both soft tissue structures such as vocal folds and muscles as well as the cartilaginous rings that support these structures.

An intricate network of regulation between the brain and the pancreas modulates hormone secretion and organ function. Dysfunction of the brain-pancreas axis occurs in disease states such as diabetes and pancreatitis. Given the delicate nature of the mouse brain, procurement for tissue and cellular analysis is facilitated by fixation by perfusion with paraformaldehyde (PFA). The brain is hardened by PFA during the preservation process, but this hardening also occurs in the pancreas, as well as the remainder of the intra-abdominal organs. This hardening makes the pancreas friable and difficult to dissect without damaging and fragmenting the organ. Additionally, this fixation may preclude the ability to perform analytic techniques such as western blot and quantitative PCR (qPCR) simultaneously. Performing a simple cross-clamping of the thoracic aorta allows for differential perfusion of organs and maximal use of limited samples from a single animal. The brain can be perfused with PFA without compromising tissue collection of the pancreas and other intra-abdominal organs. This simple maneuver allows for greater tissue collection and analysis per mouse in studies evaluating the brain-pancreas or brain-gut axis. © 2021 Wiley Periodicals LLC. Basic Protocol: Differential fixation by perfusion using aortic cross-clamp.

Complexity of lung microenvironment and changes in cellular composition during disease make it exceptionally hard to understand molecular mechanisms driving development of chronic lung diseases. Although recent advances in cell type–resolved approaches hold great promise for studying complex diseases, their implementation relies on local access to fresh tissue, as traditional tissue storage methods do not allow viable cell isolation. To overcome these hurdles, we developed a versatile workflow that allows storage of lung tissue with high viability, permits thorough sample quality check before cell isolation, and befits sequencing-based profiling. We demonstrate that cryopreservation enables isolation of multiple cell types from both healthy and diseased lungs. Basal cells from cryopreserved airways retain their differentiation ability, indicating that cellular identity is not altered by cryopreservation. Importantly, using RNA sequencing and EPIC Array, we show that gene expression and DNA methylation signatures are preserved upon cryopreservation, emphasizing the suitability of our workflow for omics profiling of lung cells. Moreover, we obtained high-quality single-cell RNA-sequencing data of cells from cryopreserved human lungs, demonstrating that cryopreservation empowers single-cell approaches. Overall, thanks to its simplicity, our workflow is well suited for prospective tissue collection by academic collaborators and biobanks, opening worldwide access to viable human tissue.

RNA sequencing (RNA-Seq) is becoming a popular method for measuring gene expression in non-model organisms, including wild populations sampled in the field. While RNA-Seq can be used to measure gene expression variation among wild-caught individuals and can yield important biological insights into organismal function, technical variables may also influence gene expression estimates. We examined the influence of multiple technical variables on estimated gene expression in a non-model fish species, the westslope cutthroat trout (Oncorhynchus clarkii lewisi), using two RNA-Seq methods: 3’ RNA-Seq and whole mRNA-Seq. We evaluated the effects of dip netting versus electrofishing, and of harvesting tissue immediately versus 5 minutes after euthanasia on estimated gene expression in blood, gill, muscle, and liver. We found higher RNA degradation in the liver compared to the other tissues. There were fewer expressed genes in blood compared to gill and muscle. We found no difference in gene expression among sampling methods or due to a delay in tissue collection. However, we detected fewer genes with 3’ RNA-Seq than with whole mRNA-Seq and found statistically significant differences in gene expression between 3’ RNA-Seq and whole mRNA-Seq. The magnitude and direction of these differences does not appear to be dependent on gene type or length. Our findings indicate that RNA-Seq is robust to the technical variables related to the field sampling techniques tested here but varies based on the tissue sampled and the RNA-Seq library used. This study advances understanding of usefulness of RNA-Seq to study gene expression variation in evolution, ecology, and conservation.

The transmission of microbial infection through tissue allografts is one of the main risks that must be controlled in tissue banks. Therefore, microbiological monitoring controls and validated protocols for the decontamination of tissues during processing have been implemented. This study is based on the evaluation of data from microbiological cultures of arteries (mainly long peripheral arteries) processed in the tissue bank of Valencia (Spain). Donors' profile, pre- and post-disinfection tissue samples were assessed. The presence of residual antibiotics in disinfected tissues was determined and the antimicrobial potential of these tissues was tested. Our overall contamination rate was 23.69%, with a disinfection rate (after antibiotic incubation) of 87.5%. Most (76.09%) of the microbial contaminants were identified as Gram positive. Arterial allografts collected from body sites affected by prior organ removal showed higher risk of contamination. Only vancomycin was detected as tissue release. The antimicrobial effect on Candida albicans was lower than that for bacterial species. Risk assessment for microbial contamination suggested the donor's skin and the environment during tissue collection as the main sources for allograft contamination. Antibiotic-disinfected arterial allografts showed antimicrobial potential.

Electron microscopy is a mainstay in the analysis of renal biopsies, where it is typically employed in a correlative fashion along with light and immunofluorescence microscopy. Despite the development of a growing armamentarium of molecular and biochemical analytic methods as well as new immunostains with a widening panel of immunoreactants, electron microscopy remains crucial to the diagnosis of a number of disorders involving the renal glomerulus, vasculature, and tubulointerstitial compartment. The number of renal biopsies continues to grow and the indications for these biopsies continue expanding together with our understanding of disease processes. Proper collection of biopsies and careful analysis of data emanating from diagnostic modalities, clinical information, imaging, gross and microscopic tissue analysis, including a wide range of ancillary studies, represent the essential paradigm for generating detailed diagnoses with clinical significance. This communication offers a guide to the pre-analytic and analytic process for renal biopsy examination, discusses diagnostic keys and pitfalls for an important category of renal diseases (immune complex disorders), and provides an introduction to a useful adjunct diagnostic method (ultrastructural immunolabeling). Renal pathologists should render expert diagnoses that guide patient management, provide prognostic information and lead to targeted new therapeutic interventions that are currently available.

An assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) has become an increasingly popular method to assess genome-wide chromatin accessibility in isolated nuclei from fresh tissues. However, many biobanks contain only snap-frozen tissue samples. While ATAC-seq has been applied to frozen brain tissues in human, its applicability in a wide variety of tissues in horse remains unclear. The Functional Annotation of Animal Genome (FAANG) project is an international collaboration aimed to provide high quality functional annotation of animal genomes. The equine FAANG initiative has generated a biobank of over 80 tissues from two reference female animals and experiments to begin to characterize tissue specificity of genome function for prioritized tissues have been performed. Due to the logistics of tissue collection and storage, extracting nuclei from a large number of tissues for ATAC-seq at the time of collection is not always practical. To assess the feasibility of using stored frozen tissues for ATAC-seq and to provide a guideline for the equine FAANG project, we compared ATAC-seq results from nuclei isolated from frozen tissue to cryopreserved nuclei (CN) isolated at the time of tissue harvest in liver, a highly cellular homogenous tissue, and lamina, a relatively acellular tissue unique to the horse. We identified 20,000–33,000 accessible chromatin regions in lamina and 22–61,000 in liver, with consistently more peaks identified using CN isolated at time of tissue collection. Our results suggest that frozen tissues are an acceptable substitute when CN are not available. For more challenging tissues such as lamina, nuclei extraction at the time of tissue collection is still preferred for optimal results. Therefore, tissue type and accessibility to intact nuclei should be considered when designing ATAC-seq experiments.

The use of post-mortem human tissue is indispensable in studies investigating alterations in metabolite levels in neurodegenerative conditions such as Alzheimer&rsquo, s disease (AD). However, variability between samples may have unknown effects on metabolite concentrations. The aim of this study was to characterize the impact of such variables. Cingulate gyrus was obtained from AD cases and controls, from three brain banks. Gas chromatography-mass spectrometry (GC-MS) was used to measure and compare the levels of 66 identifiable metabolites in these tissues to determine effects of tissue-collection variables. The effect of PMD was further investigated by analysis of rat brain cortex and cerebellum collected following post-mortem delays (PMDs) of zero to 72 h. Metabolite levels between cases and controls were not replicable across cohorts with variable age- and gender-matching, PMD, and control Braak staging. Analysis of rat tissues found significant effects of PMD on 31 of 63 identified metabolites over periods up to 72 h. PMD must be kept under 24 h for metabolomics analyses on brain tissues to yield replicable results. Tissues should also be well age- and gender-matched, and Braak stage in controls should be kept to a minimum in order to minimize the impact of these variables in influencing metabolite variability.

Although next-generation sequencing assays are routinely carried out using samples from cancer trials, the sequencing data are not always of the required quality. There is a need to evaluate the performance of tissue collection sites and provide feedback about the quality of next-generation sequencing data. This study used a modeling approach based on whole exome sequencing quality control (QC) metrics to evaluate the relative performance of sites participating in the Bristol Myers Squibb Immuno-Oncology clinical trials sample collection. We identified several events for the sample swap. Overall, most sites performed well and few showed poor performance. These findings can increase awareness of sample failure and improve the quality of samples.

Failure of rotator cuff repair surgery can be attributed to a variety of factors, including insufficient biologic environment to support healing. The subacromial bursal tissue has been shown to have a reservoir of mesenchymal stem cells and is a potential source for biologic augmentation during rotator cuff repair. We have developed a technique to capture the subacromial bursal tissue during subacromial bursectomy and then reimplant the tissue on the bursal surface of the rotator cuff tendon after rotator cuff repair. Our goal is to describe our technique of subacromial tissue collection and reimplantation that obviates the need of suturing a whole sleeve of bursal tissue while improving cell yield for rotator cuff healing.

At the heart of the phenome-to-genome approach is high throughput assays, which are liable to produce false results. This risk can be mitigated by minimizing the sample bias, specifically, recycling the same tissue specimen for both phenotypic and genotypic investigations. Therefore, our aim is to suggest a methodology of obtaining robust results from frozen specimens of compromised quality, particularly if the sample is produced in conditions with limited resources. For example, generating samples at the International Space Station (ISS) is challenging because the time and laboratory footprint allotted to a project can get expensive. In an effort to be economical with available resources, snap-frozen euthanized mice are the straightforward solution; however, this method increases the risk of temperature abuse during the thawing process at the beginning of the tissue collection. We found that prolonged immersion of snap frozen mouse carcass in 10% neutral buffered formalin at 4°C yielded minimal microscopic signs of ice crystallization and delivered tissues with histomorphology that is optimal for hematoxylin and eosin (H&E) staining and fixation on glass slides. We further optimized a method to sequester the tissue specimen from the H&E slides using an incubator shaker. Using this method, we were able to recover an optimal amount of RNA that could be used for downstream transcriptomics assays. Overall, we demonstrated a protocol that enables us to maximize scientific values from tissues collected in austere condition. Furthermore, our protocol can suggest an improvement in the spatial resolution of transcriptomic assays.

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated with an increased survival rate and greater quality of patient's life when compared to conventional dialysis. Regrettably, transplantology suffers from a lack of reliable methods for organ quality assessment. Standard diagnostic techniques are limited to macroscopic appearance inspection or invasive tissue biopsy, which do not provide comprehensive information about the graft. The proposed protocol aims to introduce solid phase microextraction (SPME) as an ideal analytical method for comprehensive metabolomics and lipidomic analysis of all low molecular compounds present in kidneys allocated for transplantation. The small size of the SPME probe enables performance of a chemical biopsy, which enables extraction of metabolites directly from the organ without any tissue collection. The minimum invasiveness of the method permits execution of multiple analyses over time: directly after organ harvesting, during its preservation, and immediately after revascularization at the recipient's body. It is hypothesized that the combination of this novel sampling method with a high-resolution mass spectrometer will allow for discrimination of a set of characteristic compounds that could serve as biological markers of graft quality and indicators of possible development of organ dysfunction.

Telomeres are used increasingly in ecology and evolution as biomarkers for ageing and environmental stress, and are typically measured from DNA extracted from nonlethally sampled blood. However, obtaining blood is not always possible in field conditions and only limited amounts can be taken from small mammals, such as bats, which moreover lack nucleated red blood cells and hence yield relatively low amounts of DNA. As telomere length can vary within species according to age and tissue, it is important to determine which tissues serve best as a representation of the organism as a whole. Here, we investigated whether wing tissue biopsies, a rapid and relatively noninvasive tissue collection method, could serve as a proxy for other tissues when measuring relative telomere length (rTL) in the Egyptian fruit bat (Rousettus aegyptiacus). Telomeres were measured from blood, brain, heart, kidney, liver lung, muscle and wing, and multiple wing biopsies were taken from the same individuals to determine intra-individual repeatability of rTL measured by using qPCR. Wing rTL correlated with rTL estimates from most tissues apart from blood. Blood rTL was not significantly correlated with rTL from any other tissue. Blood and muscle rTLs were significantly longer compared with other tissues, while lung displayed the shortest rTLs. Individual repeatability of rTL measures from wing tissue was high (>70%). Here we show the relationships between tissue telomere dynamics for the first time in a bat, and our results provide support for the use of wing tissue for rTL measurements.

Perineal talc use is associated with ovarian carcinoma in many case-control studies. Such talc may migrate to pelvic organs and regional lymph nodes, with both clinical and legal significance. Our goal was to differentiate talc in pelvic lymph nodes due to exposure, versus contamination with talc in the laboratory. We studied 22 lymph nodes from ovarian tumor patients, some of which had documented talc exposure, to quantify talc using digestion of tissue taken from paraffin blocks and scanning electron microscopy/energy dispersive X-ray analysis (SEM/EDX). Talc particles correlated significantly with surface contamination assessments using polarized light microscopy. After adjusting for surface contamination, talc burdens in nodes correlated strongly with perineal talc use. In a separate group of lymph nodes, birefringent particles within the same plane of focus as the tissues in histological sections were highly correlated with talc particles within the tissue by in situ SEM/EDX (r = 0.80; p < 0.0001). We conclude that since talc can be a surface contaminant from tissue collection/preparation, digestion measurements may be influenced by contamination. Instead, because they preserve anatomic landmarks and permit identification of particles in cells and tissues, polarized light microscopy and in situ SEM/EDX are recommended to assess talc in lymph nodes.

The complexity of the lung microenvironment together with changes in cellular composition during disease progression make it exceptionally hard to understand the molecular mechanisms leading to the development of chronic lung diseases. Although recent advances in cell type resolved and single-cell sequencing approaches hold great promise for studying complex diseases, their implementation greatly relies on local access to fresh tissue, as traditional methods to process and store tissue do not allow viable cell isolation. To overcome these hurdles, we developed a novel, versatile workflow that allows long-term storage of human lung tissue with high cell viability, permits thorough sample quality check before cell isolation, and is compatible with next generation sequencing-based profiling, including single-cell approaches. We demonstrate that cryopreservation is suitable for isolation of multiple cell types from different lung locations and is applicable to both healthy and diseased tissue, including COPD and tumor samples. Basal cells isolated from cryopreserved airways retain the ability to differentiate, indicating that cellular identity is not altered by cryopreservation. Importantly, using RNA sequencing (RNA-seq) and Illumina EPIC Array, we show that genome-wide gene expression and DNA methylation signatures are preserved upon cryopreservation, emphasizing the suitability of our workflow for -omics profiling of human lung cells. In addition, we obtained high-quality single-cell RNA sequencing data of cells isolated from cryopreserved human lung, demonstrating that cryopreservation empowers single-cell approaches. Overall, thanks to its simplicity, our cryopreservation workflow is well-suited for prospective tissue collection by academic collaborators and biobanks, opening worldwide access to human tissue.

This protocol describes low-cost methods for the collection and desiccation of plant tissues and subsequent extraction of genomic DNA. It contains modifications of previously described methods found at https://link.springer.com/book/10.1007/978-3-319-16259-1 and https://link.springer.com/chapter/10.1007/978-3-319-45021-6_14. We have tested this protocol with leaf tissue from flax, Lupinus luteus, Camelina sativa and Berberis darwinii. DNA is suitable for routine molecular biology assays such as PCR and also for next generation sequencing.

The complexity of lung microenvironment together with changes in cellular composition during disease progression hampered deeper understanding of molecular mechanisms leading to development of lung pathologies. Although recent advances in cell type resolved sequencing and single cell -omics hold great promise for studying complex diseases, their implementation greatly relies on local access to fresh tissue, as traditional tissue storage formats do not allow viable cell isolation. To overcome these hurdles, we developed a versatile workflow that allows long term storage of lung tissue with high cell viability, permits thorough sample quality check before cell isolation, and is compatible with next generation sequencing-based profiling, including single cell approaches. We demonstrate that cryopreservation is suitable for isolation of multiple cell types from different lung locations and is applicable to both healthy and tumor tissue. Basal cells isolated from cryopreserved airways retain ability to differentiate, indicating that cellular identity is not altered by cryopreservation. Importantly, using RNAseq and EPIC Array we show that genome-wide gene expression and DNA methylation signatures are preserved upon cryopreservation, emphasizing the suitability of our workflow for -omics profiling of lung cells. In addition, we obtained high quality single-cell RNAseq from cryopreserved lung tissue, demonstrating that cryopreservation empowers single-cell approaches. Overall, thanks to its simplicity, our cryopreservation workflow is well-suited for prospective tissue collection by academic collaborators and biobanks, opening worldwide access to tissue.

Made available in DSpace on 2020-12-10T19:52:11Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-01-01. Added 1 bitstream(s) on 2021-07-15T15:06:48Z : No. of bitstreams: 1 S0365-05962020000100046.pdf: 1779863 bytes, checksum: 402e59f3dcbf1af26160780023dbdeea (MD5) Fundo de Apoio a Dermatologia de Sao Paulo - FUNADERSP Background: Organoid cultures are primary cultures that maintain architectural characteristics and the relationships between cells, as well as the extracellular matrix. They are alternatives for pathophysiological or therapeutic investigation rather than animal and in vitro tests. Objective: Development of a cutaneous organoid culture model, aiming at the study of radiation-induced melanogenesis. Method: A validation study, which involved biopsies of the skin of the back of the adult ear. One sample was irradiated with different doses of UVB, UVA, or visible light (VL); the other was maintained in the dark for 72 h. The viability of the tissues was evaluated from the morphological and architectural parameters of the histology, and the expression of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, by real-time polymerase chain reaction (PCR). The radiation-induced melanin pigmentation was standardized according to the doses of each radiation and evaluated by digital image analysis (Fontana-Masson). Results: The primary skin culture was standardized at room temperature using DMEM medium. The doses of UVB, UVA, and VL (blue tight) that induced differential melanogenesis were: 166 mJ/cm(2), 1.524 J/cm(2), and 40 J/cm(2). The expression of the GAPHD constitutional gene did not differ between the sample of skin processed immediately after tissue collection and the sample cultured for 72 h in the standardized protocol. Study limitations: This was a preliminary study that evaluated only the viability and integrity of the melanogenic system, and the effect of the radiation alone. Conclusions: The standardized model maintained viable melanocytic function for 72 h at room temperature, allowing the investigation of melanogenesis induced by different forms of radiation. (C) 2019 Sociedade Brasileira de Dermatologia. Published by Elsevier Espana, S.L.U. Univ Estadual Paulista, Fac Med Botucatu, Botucatu, SP, Brazil Univ Estadual Paulista, Fac Med Botucatu, Dept Dermatol & Radiotherapy, Botucatu, SP, Brazil Univ Estadual Paulista, Fac Med Botucatu, Dept Dermatol & Radiotherapy, Grad Program Pathol, Botucatu, SP, Brazil Univ Estadual Paulista, Fac Med Botucatu, Dept Pathol, Botucatu, SP, Brazil Univ Estadual Paulista, Fac Med Botucatu, Botucatu, SP, Brazil Univ Estadual Paulista, Fac Med Botucatu, Dept Dermatol & Radiotherapy, Botucatu, SP, Brazil Univ Estadual Paulista, Fac Med Botucatu, Dept Dermatol & Radiotherapy, Grad Program Pathol, Botucatu, SP, Brazil Univ Estadual Paulista, Fac Med Botucatu, Dept Pathol, Botucatu, SP, Brazil

The Functional Annotation of Animal Genomes (FAANG) project aims to identify genomic regulatory elements in both sexes across multiple stages of development in domesticated animals. This study represents the first stage of the FAANG project for the horse, Equus caballus. A biobank of 80 tissue samples, two cell lines and six body fluids was created from two adult Thoroughbred mares. Ante-mortem assessments included full physical examinations, lameness, ophthalmologic and neurologic evaluations. Complete blood counts and serum biochemistries were also performed. At necropsy, in addition to tissue samples, aliquots of serum, ethylenediaminetetraacetic acid (EDTA) plasma, heparinized plasma, cerebrospinal fluid, synovial fluid, urine and microbiome samples from all regions of the gastrointestinal and urogenital tracts were collected. Epidermal keratinocytes and dermal fibroblasts were cultured from skin samples. All tissues were grossly and histologically evaluated by a board-certified veterinary pathologist. The results of the clinical and pathological evaluations identified subclinical eosinophilic and lymphocytic infiltration throughout the length of the gastrointestinal tract as well as a mild clinical lameness in both animals. Each sample was cryo-preserved in multiple ways, and nuclei were extracted from selected tissues. These samples represent the first published systemically healthy equine-specific biobank with extensive clinical phenotyping ante- and post-mortem. The tissues in the biobank are intended for community-wide use in the functional annotation of the equine genome. The use of the biobank will improve the quality of the reference annotation and allow all equine researchers to elucidate unknown genomic and epigenomic causes of disease.

The objectives of this study were to develop a methodology for biopsying the rumen and colon of young dairy calves and to collect suitable quality tissue samples for microscopic and gene expression analysis. Six Holstein dairy bull calves (45.0 ± 1.5 kg birth weight) were ruminally cannulated during the second week of life and weaned at the end of wk 6. Ruminal and colon tissue samples were collected at the end of wk 5, 6, 7, 8, and 12. Calves were not sedated but were restrained in a chute for sampling. The endoscope (100 cm length, 9.8 mm diameter) was introduced through the rumen cannula to harvest ruminal tissue. Endoscopic biopsies of the rumen with endoscopic biopsy forceps were unsuccessful 85% of the time because they were unable to shear the ruminal tissue. Thereafter, an Allis clamp was used to retrieve the blind sac through the rumen cannula to perform direct tissue biopsying with surgical scissors. To biopsy the colon, the lubricated distal tip of an endoscope was slowly inserted into the calf's anus. A total of 6 colon tissue samples (12.6 ± 0.74 mg) were collected per calf per time point from the distal colon 30 to 40 cm from the calf's anus using endoscopic biopsy forceps, which were inserted through the instrument channel. A new forcep was used between sites and calves. Between calves, the outside of the endoscope was washed with 4% chlorohexidine and rinsed with water and the instrument channel was washed with distilled water and 70% ethanol. Colon and ruminal samples were processed for histological measurements, and RNA was isolated and sequenced. High-quality RNA (RNA integrity number 8.8 ± 0.08) was collected from samples, and light and electron microscopy was performed on samples. In conclusion, endoscopic biopsying can be used for tissue harvest in the colon of young calves. However, it was found that collecting ruminal tissue by retracting the rumen from the cannula and taking samples with surgical scissors was more successful than an endoscopic biopsy. This method allows for tissue collection of the same animal throughout time, which can help the research community investigate the effect of weaning regimens, feed rations, and age on the structure and function of the gastrointestinal tract.

Examining genetic diversity, population structure, and geographic distribution has become an important part in the conservation strategies of endangered populations. However, these studies rely on tissue samples collection for DNA analysis which may be problematic for species with a sensitive conservation status. Partial fin-clipping has been employed for tissue collection, due to the increased popularity of DNA-based analysis for ecological and fisheries studies, however, fin clipping can potentially affect behavior, swimming performance, predator avoidance and the ability to find and capture prey. This study aimed to test the effects of filament clipping using captive breed seahorses (Hippocampus guttulatus) as an alternative tool for tissue sample. Clipped filaments regrew on average 0.05 ± 0.02 mm/day for 3 months and no mortality or disease was observed during the experiment. Filaments provided enough tissue for DNA analysis. This study provided valuable information regarding a new sampling technique that does not impair the seahorse locomotion. This methodology may be used in population’s genetic studies of other species that have skin filaments as a morphological trait.

Purpose of Review Studying and developing preanalytical tools and technologies for the purpose of obtaining high-quality samples for histological assays is a growing field. Currently, there does not exist a standard practice for collecting, fixing, and monitoring these precious samples. There has been some advancement in standardizing collection for the highest profile tumor types, such as breast, where HER2 testing drives therapeutic decisions. This review examines the area of tissue collection, transport, and monitoring of formalin diffusion and details a prototype system that could be used to help standardize tissue collection efforts. Recent Findings We have surveyed recent primary literature sources and conducted several site visits to understand the most error-prone processes in histology laboratories. This effort identified errors that resulted from sample collection techniques and subsequent transport delays from the operating room (OR) to the histology laboratories. We have therefore devised a prototype sample collection and transport concept. The system consists of a custom data logger and cold transport box and takes advantage of a novel cold + warm (named 2 + 2) fixation method. Summary This review highlights the beneficial aspects of standardizing tissue collection, fixation, and monitoring. In addition, a prototype system is introduced that could help standardize these processes and is compatible with use directly in the OR and from remote sites.

Objective To determine how frequently and effectively products of conception can be obtained among women pursuing medical management of early pregnancy loss. Methods This pilot study was conducted to assess products of conception recovery outcomes for participants opting for medical management compared with women opting for surgical aspiration A tissue-collection kit was provided to women opting for medical management. Outcome measures included successful collection of products of conception, quantity and integrity of DNA, and participant satisfaction with the process. Results Tissue was collected from 19 of 22 participants in the medical management group (84%) and 39 participants (100%) in the surgical management group (P = .02). DNA yield and integrity were similar among both groups (P = .03 and P = .003, respectively). Participants in the medical group reported a high comfort level with the kit and the process of tissue collection. Conclusions Medical management of a missed abortion followed by patient-controlled collection of products of conception for subsequent cytogenetic analysis is well tolerated and highly effective. This methodology may reduce the need for surgical management, empower women to have more agency in their medical decisions, and increase access to genetic testing.

application/pdf, Activin is secreted from equine uterine glands and plays important roles in establishment and maintenance of pregnancy in mares. This study aimed to localize activin receptors (ActRs) IA/B and IIA/B using immunohistochemistry in the uteroplacental tissues of seven pregnant Thoroughbred mares. At the time of tissue collection, the mares were at the following days of pregnancy: 88, 120, 161, 269, 290, 313, and 335 days. We fixed the uteroplacental tissues in 4% paraformaldehyde and obtained serial sections that were subsequently stained for analysis. All four isoforms of ActR were expressed in the uteroplacental tissues, including the endometrial epithelium, uterine glands, trophoblasts, and myometrium, throughout pregnancy. Our results suggested the potential role of activin in the uteroplacental tissues. © 2018 Japanese Society of Equine Science.