Standardized protocol for collecting bee samples to generate molecular data

Biobanking is the process of storing high quality human biospecimens alongside linked clinical data, for research purposes. The aim is to identify novel biomarkers with prognostic or diagnostic significance. However, the challenges implicit in the collection and storage of human tissue for research have curtailed the impact of this technique to date. This paper aims to summarise the challenges faced by biobanking within the ENT specialty in the UK, and to present protocols used for the routine collection, freezing and storage of tissue specimens at the Royal National Throat, Nose and Ear Hospital. These protocols could be used to guide other ENT departments (in the UK and worldwide) wishing to initiate the routine collection and storage of tissue samples. Their publication could also help to establish basic standards and ensure consistency in ENT tissue storage. Interviews conducted with industry experts, and a literature review of 'best practice' in biobanking. The ENT specialty must stay abreast of progress in human tissue research in order to ensure the best possible management of its patients. Our protocol for the routine banking of ENT tissue at the Royal National Throat, Nose and Ear Hospital could be used as a template for other ENT departments (in the UK and worldwide) to encourage widespread implementation of high quality tissue banking.

The increasingly complex subclassification of tumors based on clinical and molecular characteristics, as well as the large sample sizes needed for clinical trials, pose new challenges to the recruitment of patients for studies. Currently, less than 10% of all cancer patients are enrolled in clinical trials, limiting their external validity, but also leaving room for improvement in the recruitment of patients. Clinical trials are often complicated by low inclusion rates, inadequate data collection, high non-completion rates and costs, and often provide insufficient data for post-hoc subgroup analyses. We believe that a prospective observational cohort study can provide standardized and validated collection of clinical data, tissue and blood samples and patient-reported outcome measures, and can improve patient recruitment for clinical trials. The Prospective Dutch ColoRectal Cancer cohort is a prospective multidisciplinary nation-wide observational cohort study in the Netherlands, a country with a yearly colorectal cancer incidence of 15,000 patients. The goal of the study is to facilitate future basic, translational and clinical research in the field of colorectal cancer for both national and international research groups with short or absent inclusion periods, even for studies with stringent inclusion criteria. All patients > 18 years with histologically proven colorectal cancer are asked to participate. Four essential features characterize the study: 1) a patient-centered informed-consent approach; 2) a framework to systematically collect long-term clinical follow-up and patient-reported outcomes; 3) an infrastructure for the standardized collection and storage of tissue and blood samples and 4) efficient recruitment for clinical trials which is facilitated by the innovative cohort multiple randomized controlled trial (cmRCT) design. The cmRCT design allows patients to participate in multiple non-conflicting clinical trials. We believe that the key to the development of a sustainable cohort study is the collaboration with existing organizations using their expertise, and preventing duplicate data entry and unnecessary costs. To these ends, close collaborations with other national initiatives, like the national cancer registry (hosted by IKNL), the national pathology registry PALGA, and the national biobanking infrastructure BBMRI-NL have been established. An effective governance structure to secure the privacy of patients and hospitals, and to facilitate research is needed. This study will provide long-term clinical data, tissue and blood samples, and patient-reported outcome measures collected under a broad informed consent, of a large cohort of patients with colorectal cancer. The available data and tissue will facilitate basic, translational and clinical research. Furthermore, the design of this cohort study can be used as an example for other tumor types. Citation Format: Robert R.J. Coebergh van den Braak, Geraldine R. Vink, Martijn G.H. van Oijen, Mirre E. de Noo, Sophie A. Kurk, Maarten J.P. Burbach, Alice M. Couwenberg, Anne M. May, Helena M. Verkooijen, Gerrit A. Meijer, Miriam Koopman. The prospective Dutch colorectal cancer cohort: A prospective nation-wide observational cohort study. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1827.

The heterogeneity within individual distinct cancer types in terms of behaviour, response to therapy and prognosis is well recognized. A major goal of translational research projects has therefore been to define clinically significant subgroups of individual tumour types by analysis of mRNA as well as protein expression. An essential premise of such investigations is that expression of these key molecules is a true reflection of conditions present within the neoplastic cells in vivo. The aim was to investigate the effect of methods of tissue handling and storage on expression of mRNA. mRNA expression in 60 biopsy samples obtained from 10 patients with colorectal tumours was examined. The mRNA expression profile and the level of expression of specific mRNA species were significantly affected by the procedures used for collection and storage of tissue samples. Significant variation in the level of expression (both increased and decreased) of transcripts was detectable after 15 min, and by 120 min there was a fourfold increase in the number of genes with a more than twofold change in the level of expression. Reliable interpretation of results of gene expression at the mRNA level requires standardized protocols for tissue procurement.

In recent studies, the life span of stroke-prone spontaneously hypertensive (SHRSP) rats was altered by a variety of dietary fats. It was relatively shorter in rats fed canola oil as the sole source of fat. The present study was performed to find out whether the fatty acid profile and the high content of sulfur compounds in canola oil could modulate the life span of SHRSP rats. SHRSP rats (47 d old, n = 23/group) were matched by body weight and systolic blood pressure and fed semipurified diets containing 10% canola oil, high-palmitic canola oil, low-sulfur canola oil, soybean oil, high-oleic safflower oil, a fat blend that mimicked the fatty acid composition of canola oil, or a fat blend high in saturated fatty acids. A 1% sodium chloride solution was used as drinking water to induce hypertension. After consuming the diets for 37 d, five rats from each dietary group were killed for collection of blood and tissue samples for biochemical analysis. The 18 remaining animals from each group were used for determining their life span. The mean survival time of SHRSP rats fed canola oil (87.4+/-4.0 d) was not significantly different (P > 0.05) from those fed low-sulfur canola oil (89.7+/-8.5 d), suggesting that content of sulfur in canola oil has no effect on the life span of SHRSP rats. The SHRSP rats fed the noncanola oil-based diets lived longer (mean survival time difference was 6-13 d, P < 0.05) than those fed canola and low-sulfur canola oils. No marked differences in the survival times were observed among the noncanola oil-based groups. The fatty acid composition of the dietary oils and of red blood cells and liver of SHRSP rats killed after 37 d of treatment showed no relationship with the survival times. These results suggest that the fatty acid profile of vegetable oils plays no important role on the life span of SHRSP rat. However, phytosterols in the dietary oils and in liver and brain were inversely correlated with the mean survival times,indicating that the differential effects of vegetable oils might be ascribed, at least partly, to their different phytosterol contents.

Chromosome instability and the FAMMM syndrome

The consumption of high-fat diets is a global problem. The use of micronutrients, such as calcium and vitamin D, has positive effects on glucose metabolism during pregnancy, reproductive performance, and embryofetal development. This study aims to evaluate the effects of a mixture of micronutrients on glucose metabolism and perinatal outcomes in rats exposed to a high-fat diet (HFD). The female rats received a standard diet (SD) or high-fat diet (HFD) from weaning to the end of pregnancy. The pregnant rats were treated with the micronutrient mixture (Mix) only during pregnancy, and were distributed into 4 groups: SD, SD+Mix, HFD, and HFD+Mix. At the end of pregnancy, the rats were euthanized for collection of maternal adipose tissue, maternal and fetal blood samples, and placental samples. The HFD dams presented a higher number of fetuses with low weight at birth compared to the SD groups. Fetuses from SD+Mix and HFD+Mix had higher serum concentrations of vitamin D compared to the control respective groups. The HFD+mix group had higher maternal serum concentrations of vitamin D and increased rate of fetal viability, lower maternal visceral fat weight and lower percentage of embryonic deaths compared to the HFD group. Thus, the Mix treatment causes benefits to mothers and offspring exposed to HFD intake because of improved maternal glycemia, lower adiposity index, increased serum vitamin D, and embryofetal viability. This study shows the importance of prioritizing a healthy diet to achieve maternal physical well-being and enable the healthy development of offspring.

Collection of tissue and subsequent isolation of genomic DNA from mature tree species often proves difficult. DNA extraction from needles, leaves, or buds is recommended in many protocols. Collecting these tissues from mature trees generally requires the use of firearms or climbing if sampling is to be nondestructive. As a result, sample collection is a major expense of many tree-based projects. Tree (and plant) tissues generally contain large amounts of polysaccharides and phenolic compounds that are difficult to separate from DNA. Many methods aim to overcom these problems, with most involving extraction in buffers containing the nonionic detergent cetyltrimethyl-ammonium bromide (CTAB), followed by numerous steps to clean contaminants from the DNA, using organic solvents and differential salt precipitation. These steps are time-consuming, such that isolation of DNA becomes the bottleneck in many molecular studies. This paper presents a new, efficient, cambium collection method for tree species and a DNA extraction protocol based on that of Doyle and Doyle (1987), with follow-up purification using the Wizard nuclei lysis and protein precipitation solutions (Promega). Results show a significant improvement in yield and DNA purity compared with other published methods, with consistently high yields of pure genomic DNA and high sample throughput. The relatively low cost per extraction, no requirement for use of liquid nitrogen, no requirement for freezer storage, and long-term sample stability after collection are important additional benefits.

ObjectiveTo harmonize standard operating procedures (SOPs) and standardize the recording of associated data for collection, processing, and storage of human tissues relevant to endometriosis.DesignAn international collaboration involving 34 clinical/academic centers and three industry collaborators from 16 countries on five continents.SettingIn 2013, two workshops were conducted followed by global consultation, bringing together 54 leaders in endometriosis research and sample processing from around the world.Patient(s)None.Intervention(s)Consensus SOPs were based on: 1) systematic comparison of SOPs from 24 global centers collecting tissue samples from women with and without endometriosis on a medium or large scale (publication on >100 cases); 2) literature evidence where available, or consultation with laboratory experts otherwise; and 3) several global consultation rounds.Main Outcome Measure(s)Standard recommended and minimum required SOPs for tissue collection, processing, and storage in endometriosis research.Result(s)We developed “recommended standard” and “minimum required” SOPs for the collection, processing, and storage of ectopic and eutopic endometrium, peritoneum, and myometrium, and a biospecimen data collection form necessary for interpretation of sample-derived results.Conclusion(s)The EPHect SOPs allow endometriosis research centers to decrease variability in tissue-based results, facilitating between-center comparisons and collaborations. The procedures are also relevant to research into other gynecologic conditions involving endometrium, myometrium, and peritoneum. The consensus SOPs are based on the best available evidence; areas with limited evidence are identified as requiring further pilot studies. The SOPs will be reviewed based on investigator feedback and through systematic triannual follow-up. Updated versions will be made available at: http://endometriosisfoundation.org/ephect.

Brain tissue obtained at autopsy has been used in research for non-oncologic disorders. However, to the best of the authors' knowledge, this tool has never been systematically used in large investigational studies for cancer. A prospective, multicenter study was conducted to assess the feasibility of tissue collection at autopsy and its suitability for molecular analyses in children with diffuse intrinsic pontine glioma. Tumor tissue was collected at the time of diagnosis, if clinically indicated, or at autopsy. Normal brain tissue was also collected at autopsy. The integrity of DNA and RNA was evaluated in all samples. Logistic data regarding autopsies were recorded. The feasibility of tissue collection at autopsy was assessed for patients treated at a single institution over a 43-month period. Tumor samples were collected at the time of diagnosis (n = 3) or at autopsy (n = 38) at 29 centers across the United States; samples were obtained at diagnosis and autopsy in 2 cases. The median interval from death to autopsy was 7.7 hours. DNA and RNA with minimal or partial degradation, which were suitable for genome-wide analysis, were obtained from 100% and 63% of tumor samples, respectively. At the coordinating institution, approximately 40% of parents consented to autopsy and 40% declined. During the study period, 12 autopsies were performed on patients who did not receive therapy at the coordinating center. Multicenter, biological studies based on tissue obtained at autopsy appear to be feasible in children with brain cancer. The current experience established a new paradigm for brain tissue collection, which may increase the potential for research studies in patients with cancer.

Advancements in personalized medicine require secure, transparent, and privacy-preserving genomic data management systems. This study proposes a novel hybrid blockchain-based genomic data model integrating Self-Sovereign Identity (SSI), Decentralized Identifiers (DIDs), Verifiable Credentials (VCs), and decentralized storage Interplanetary File System (IPFS) to enable secure, privacy-compliant, patient-controlled genomic data exchange. The model leverages Polygon Proof-of-Stake (PoS) blockchain by deploying a smart contract that enforces fully access control functions applied on 100 samples of synthetic genomic data, ensuring only authorized researchers with valid DIDs and VCs can retrieve genomic data. Later, we performed five tests to evaluate our model performance. Security evaluations confirmed 100% data integrity validation through SHA-256 hash validation on-chain, ensuring tamper-proof genomic data storage. Unauthorized access attempts resulted in zero successful breaches, demonstrating the robustness of SSI-based authentication by showing revoked access. Additionally, IPFS data availability testing confirmed reliable and decentralized data retrieval through the Content Identifier (CID) on-chain. The model's access revocation mechanism enabled real-time patient control over genomic data access, ensuring compliance with GDPR privacy regulations. The proposed model provides a scalable, secure, and privacy-compliant solution for genomic data sharing in precision medicine, empowering patients with full control over their genetic data while facilitating researchers to trustworthy, decentralized data useability for precision research.

The purpose of this study was to compare bacteriological urinalysis findings using 3 urinary sample collection methods (clean stoma catheterization, urine dripping from the stoma, urine collected from the clean urostomy pouch) in ileal conduit urinary diversion patients. Randomized controlled trial. Twenty-seven patients with ileal conduit urinary diversion from an outpatient urology clinic were enrolled; 9 patients were seen twice, for a total of 36 subjects and comparisons. Data were collected during a clinic visit by a trained research nurse. Patients were randomized into 2 groups: group A had the first urine sample collected by clean stoma catheterization, followed by sample collection by urine dripping from the stoma; group B had the first urine sample collection by urine dripping from the stoma, followed by sample collected by clean stoma catheterization. All patients had a third urine sample collected from a factory-clean urostomy pouch. Bacteriological urinalysis findings were compared among methods. Descriptive analyses were summarized using mean, percentage, and frequency. The mean ages of the patients between the groups were compared with the t test. Other between-group comparisons were performed using the Fisher exact test. Urinary culture finding differences among the same patients were evaluated using the McNemar test. Sensitivity and specificity of the different urine sample collection methods were calculated assuming urine sample collection by catheterization as a reference method. Uropathogen bacteria were detected in urinary culture in 16 of 36 samples (44%) collected by clean stoma catheterization, 15 of 36 samples (42%) collected by urine dripping directly from the stoma, and 13 of 35 samples (37%) collected from the clean urostomy pouch. Significant differences among the urine collection methods were not detected. Assuming catheterization as the most reliable method of sample collection, the sensitivity and specificity of the urine dripping from stoma collection method were 81.3% and 90.0%, respectively. The sensitivity and specificity of the urostomy pouch collection method were 73.3% and 90.0%, respectively. Among the same patients, there were no significant differences in the incidence of uropathogen bacteria when clean stoma catheterization was compared with urine dripping from the stoma and urostomy pouch methods. This study provides clinically relevant information regarding urine collection methods in ileal conduit patients. Urinary sample collection by urine dripping directly from the stoma or collected from a clean urostomy pouch provided similar uropathogen bacteria findings compared with sample collection by clean stoma catheterization.

No standardized method has yet been established for evaluating airway mucosal aberrancies associated with chronic obstructive pulmonary disease (COPD) or chronic bronchitis (CB). While goblet cell hyperplasia (GCH) is an established pathognomonic hallmark of the CB disease process, no standardized method exists for acquiring mucosal biopsies and assessing morphologic airway mucosa alterations. Additionally, the impacts from interventions targeting the airway mucosa are not well defined. In this context, a reliable and robust measure for assessing airway mucosa at baseline and subsequent to an intervention is critical for characterizing treatment-related changes. Can standardizing airway biopsy tissue collection and histopathological assessment methods generate a robust and repeatable measure to assess airway mucosa tissue characteristics in the setting of COPD/CB? Initial tissue collection and histological assessment methods were designed by integrating various aspects from previously published evaluations, applied to an initial tissue sample cohort, and then iteratively refined by independent pathologists. A standardized metric for histologic airway mucosa assessments was developed that specified tissue collection methods, including re-sampling airways at multiple time points to enable evaluation of treatment-related effects by incorporating scores for GCH, eosinophilia, and chronic inflammation, and the degree of GCH heterogeneity present within each sample. This multi-center study generated a robust, reproducible approach for assessing airway mucosa aberrancies in the setting of COPD/CB. The iterative approach established consistent tissue specimen recovery and a granular scoring matrix that enabled quantitative scoring of the various tissue findings with substantial histopathologic interrater reliability. ClinicalTrials.gov; NCT03107494, NCT04677465; URL: www. gov. Australian New Zealand Clinical Trials Registry (ANZCTR); ACTRN12617000330347; URL: anzctr.org.au.

Focused ultrasound (FUS) is a powerful emerging tool for non-invasive, non-ionizing targeted destruction of tumors. The last two decades have seen a growing body of preclinical and clinical literature supporting...

Psychiatry has traditionally focused on the study of neurons and neurotransmitter physiology in the pathophysiology and treatment of psychiatric disorders. A growing literature highlights REDOX imbalance (a state in which demand for antioxidants surpasses their bioavailability) as a common pathophysiology for a diverse array of brain conditions (e.g., trichotillomania, schizophrenia, autism, Parkinson’s disease). REDOX imbalance is typically measured via plasma glutathione, as glutathione is critical to the adaptive antioxidant response in the brain. Accordingly, glutathione, its precursors, and/or metabolites serve as biomarkers of disease risk, therapeutic targets, and measures of treatment response. However, as with any emerging field, there are currently several different methods for collection, processing, storage, and calculation of summary measures of plasma glutathione metabolism, within and between preclinical and clinical research. The lack of evidence-based best-practice methodology hampers reproducibility (preclinical or clinical), and translation (between preclinical and clinical work). To address this methodological need, here we used a repeated measures within-subject design to investigate how sample preparation (type of anticoagulant used during blood collection, deproteinization status, and storage temperature) affects plasma glutathione levels. Accordingly, we collected whole blood from mice (N = 13), and then, using a commercially available kit, quantified glutathione in plasma stored in four different ways. Presuming that these preparation conditions and post-processing calculations are unimportant, we would expect to see no difference in glutathione levels and summary measures from the same sample. However, we found each of these variables to significantly alter quantified glutathione levels. Accordingly, we propose a vital, gold-standard methodology for both sample collection, processing, and storage of plasma used for glutathione quantification and for summary calculations of glutathione that can be used preclinically and clinically, thus yielding more streamlined translation.

BackgroundThis article presents the methodology for tissue sample collection in Trans-CHHiP, the main translational study within the CHHiP (Conventional or Hypofractionated High dose intensity modulated radiotherapy in Prostate cancer, ISRCTN 97182923) trial. The CHHiP trial randomised 3216 men with localised prostate cancer to 3 different radiotherapy fractionation schedules. Trans-CHHiP aims to identify biomarkers of fraction sensitivity. MethodsWe outline the process of tissue collection, including central review by a study-specific specialist uropathologist and comparison of the centrally-assigned Gleason grade group with that assigned by the recruiting-centre pathologist. Results2047 patients provided tissue from 107 pathology departments between August 2012 and April 2014. A highly motivated Clinical Trials Unit chasing samples and a central Trans-CHHiP group that regularly reviewed progress were important for successful sample collection. Agreement in Gleason grade group assigned by the recruiting centre pathologist and the central study-specific uropathologist occurred in 886 out of 1854 (47.8%) cases. Key lessons learned were the need for prospective consent for tissue collection when recruiting patients to the main trial, and the importance of Material Transfer Agreement (MTA) integration into the initial trial site agreement. ConclusionsThis methodology enabled collection of 2047 patient samples from a large randomised radiotherapy trial. Central pathological review is important to minimise subjectivity in Gleason grade grouping and the impact of grade shift.