RESUMO
International consensus supports the development of standardized protocols for measured glomerular filtration rate (mGFR) to facilitate the integration of mGFR testing in both clinical and research settings. To this end, the European Kidney Function Consortium convened an international group of experts with relevant experience in mGFR. The working group performed an extensive literature search to inform the development of recommendations for mGFR determination using 1-compartment plasma clearance models and iohexol as the exogenous filtration marker. Iohexol was selected as it is non-radio labeled, inexpensive, and safe, can be assayed at a central laboratory, and the other commonly used non-radio-labeled tracers have been (inulin) or are soon to be (iothalamate) discontinued. A plasma clearance model was selected over urine clearance as it requires no urine collection. A 1 compartment was preferred to 2 compartments as it requires fewer samples. The recommendations are based on published evidence complemented by expert opinion. The consensus paper covers practical advice for patients and health professionals, preparation, administration, and safety aspects of iohexol, laboratory analysis, blood sample collection and sampling times using both multiple and single-sample protocols, description of the mGFR mathematical calculations, as well as implementation strategies. Supplementary materials include patient and provider information sheets, standard operating procedures, a study protocol template, and support for mGFR calculation.
Assuntos
Consenso , Meios de Contraste , Taxa de Filtração Glomerular , Iohexol , Rim , Adulto , Humanos , Meios de Contraste/efeitos adversos , Meios de Contraste/farmacocinética , Meios de Contraste/administração & dosagem , Europa (Continente) , Iohexol/farmacocinética , Iohexol/análise , Taxa de Depuração Metabólica , Modelos BiológicosRESUMO
BACKGROUND AIMS: Human umbilical cord-derived mesenchymal stromal cells (hUC-MSCs) are increasingly used in research and therapy. To obtain hUC-MSCs, a diversity of isolation and expansion methods are applied. Here, we report on a robust and standardized method for hUC-MSC isolation and expansion. METHODS: Using 90 hUC donors, we compared and optimized critical variables during each phase of the multi-step procedure involving UC collection, processing, MSC isolation, expansion and characterization. Furthermore, we assessed the effect of donor-to-donor variability regarding UC morphology and donor attributes on hUC-MSC characteristics. RESULTS: We demonstrated robustness of our method across 90 UC donors at each step of the procedure. With our method, UCs can be collected up to 6 h after birth, and UC-processing can be initiated up to 48 h after collection without impacting on hUC-MSC characteristics. The removal of blood vessels before explant cultures improved hUC-MSC purity. Expansion in Minimum essential medium α supplemented with human platelet lysate increased reproducibility of the expansion rate and MSC characteristics as compared with Dulbecco's Modified Eagle's Medium supplemented with fetal bovine serum. The isolated hUC-MSCs showed a purity of â¼98.9%, a viability of >97% and a high proliferative capacity. Trilineage differentiation capacity of hUC-MSCs was reduced as compared with bone marrow-derived MSCs. Functional assays indicated that the hUC-MSCs were able to inhibit T-cell proliferation demonstrating their immune-modulatory capacity. CONCLUSIONS: We present a robust and standardized method to isolate and expand hUC-MSCs, minimizing technical variability and thereby lay a foundation to advance reliability and comparability of results obtained from different donors and different studies.
Assuntos
Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais , Humanos , Reprodutibilidade dos Testes , Cordão Umbilical , Diferenciação Celular , Proliferação de CélulasRESUMO
Hyperpolarized (HP) 129 Xe MRI uniquely images pulmonary ventilation, gas exchange, and terminal airway morphology rapidly and safely, providing novel information not possible using conventional imaging modalities or pulmonary function tests. As such, there is mounting interest in expanding the use of biomarkers derived from HP 129 Xe MRI as outcome measures in multi-site clinical trials across a range of pulmonary disorders. Until recently, HP 129 Xe MRI techniques have been developed largely independently at a limited number of academic centers, without harmonizing acquisition strategies. To promote uniformity and adoption of HP 129 Xe MRI more widely in translational research, multi-site trials, and ultimately clinical practice, this position paper from the 129 Xe MRI Clinical Trials Consortium (https://cpir.cchmc.org/XeMRICTC) recommends standard protocols to harmonize methods for image acquisition in HP 129 Xe MRI. Recommendations are described for the most common HP gas MRI techniques-calibration, ventilation, alveolar-airspace size, and gas exchange-across MRI scanner manufacturers most used for this application. Moreover, recommendations are described for 129 Xe dose volumes and breath-hold standardization to further foster consistency of imaging studies. The intention is that sites with HP 129 Xe MRI capabilities can readily implement these methods to obtain consistent high-quality images that provide regional insight into lung structure and function. While this document represents consensus at a snapshot in time, a roadmap for technical developments is provided that will further increase image quality and efficiency. These standardized dosing and imaging protocols will facilitate the wider adoption of HP 129 Xe MRI for multi-site pulmonary research.
Assuntos
Pulmão , Isótopos de Xenônio , Pulmão/diagnóstico por imagem , Imageamento por Ressonância Magnética , Estudos Multicêntricos como Assunto , Ventilação Pulmonar , RespiraçãoRESUMO
Metabolic profiling by 1H NMR spectroscopy is an underutilized technology in salivary research, although preliminary studies have identified promising results in multiple fields (diagnostics, nutrition, sports physiology). Translation of preliminary findings into validated, clinically approved knowledge is hindered by variability in protocol for the collection, storage, preparation, and analysis of saliva. This study aims to evaluate the effects of differing sample pretreatments on the 1H NMR metabolic profile of saliva. Protocol considerations are highly varied in the current literature base, including centrifugation, freeze-thaw cycles, and different NMR quantification methods. Our findings suggest that the 1H NMR metabolite profile of saliva is resilient to any change resulting from freezing, including freezing of saliva prior to centrifuging. However, centrifugation was necessary to remove an unidentified broad peak between 1.24 and 1.3 ppm, the intensity of which correlated strongly with saliva cellular content. This peak obscured the methyl peak from lactate and significantly affected quantification. Metabolite quantification was similar for saliva centrifuged between 750 g to 15â¯000 g. Quantification of salivary metabolites was similar whether quantified using internal phosphate-buffered sodium trimethylsilyl-[2,2,3,3-2H4]-propionate (TSP) or external TSP in a coaxial NMR tube placed inside the NMR tube containing the saliva sample. Our results suggest that the existing literature on salivary 1H NMR will not have been adversely affected by variations of the common protocol; however, use of TSP as an internal standard without a buffered medium appears to affect metabolite quantification, notably for acetate and methanol. We include protocol recommendations to facilitate future NMR-based studies of saliva.
Assuntos
Espectroscopia de Prótons por Ressonância Magnética/métodos , Projetos de Pesquisa/normas , Saliva/química , Manejo de Espécimes/normas , Soluções Tampão , Centrifugação , Congelamento , Humanos , Metaboloma , Metabolômica , Espectroscopia de Prótons por Ressonância Magnética/normas , Padrões de Referência , Saliva/metabolismo , Manejo de Espécimes/métodos , Compostos de TrimetilsililRESUMO
AIM: To evaluate and quantify the intervention fidelity of a symptom management protocol through implementation of a scorecard, using an exemplar study of caregiver-delivered reflexology for people with breast cancer. BACKGROUND: Studies on caregiver-delivered symptom management interventions seldom include adequate information on protocol fidelity, contributing to potentially suboptimal provision of the therapeutic intervention, hindering reproducibility and generalizability of the results. DESIGN: Fidelity assessment of a 4-week intervention protocol in a randomized controlled trial (RCT) with data collection between 2012 - 2016. METHODS: The National Institutes of Health Behaviour Change Consortium (NIH-BCC) conceptual model for intervention fidelity guided the study. The five NIH-BCC fidelity elements are: (1) dose; (2) provider training; (3) intervention delivery; (4) intervention receipt; and (5) enactment. To illustrate the elements, an intervention protocol was deconstructed and each element quantified using a newly developed fidelity scorecard. RESULTS: Mean scores and frequency distributions were derived for the scorecard elements. For dose, the mean number of sessions was 4·4, 96% used the correct intervention duration and 29% had 4 weeks with at least one session. Provider training was achieved at 80% of the maximum score, intervention delivery was 96%, intervention receipt was 99% and enactment indicated moderate adoption at 3·8 sessions per patient. The sample mean score was 15·4 out of 16, indicating the high overall fidelity. CONCLUSION: Research findings that include description of how fidelity is both addressed and evaluated are necessary for clinical translation. Clinicians can confidently recommend symptom management strategies to patients and caregivers when fidelity standards are explicitly reported and measured.
Assuntos
Neoplasias da Mama/terapia , Cuidadores/normas , Atenção à Saúde/normas , Adolescente , Adulto , Idoso , Cuidadores/educação , Feminino , Humanos , Massagem/educação , Massagem/métodos , Pessoa de Meia-Idade , Adulto JovemRESUMO
Protocol standardization and sharing are crucial for reproducibility in life sciences. In spite of numerous efforts for standardized protocol description, adherence to these standards in literature remains largely inconsistent. Curation of protocols are especially challenging due to the labor intensive process, requiring expert domain knowledge of each experimental procedure. Recent advancements in Large Language Models (LLMs) offer a promising solution to interpret and curate knowledge from complex scientific literature. In this work, we develop ProtoCode, a tool leveraging fine-tune LLMs to curate protocols into intermediate representation formats which can be interpretable by both human and machine interfaces. Our proof-of-concept, focused on polymerase chain reaction (PCR) protocols, retrieves information from PCR protocols at an accuracy ranging 69-100 % depending on the information content. In all tested protocols, we demonstrate that ProtoCode successfully converts literature-based protocols into correct operational files for multiple thermal cycler systems. In conclusion, ProtoCode can alleviate labor intensive curation and standardization of life science protocols to enhance research reproducibility by providing a reliable, automated means to process and standardize protocols. ProtoCode is freely available as a web server at https://curation.taxila.io/ProtoCode/.
Assuntos
Reação em Cadeia da Polimerase , Reação em Cadeia da Polimerase/métodos , Humanos , Software , Reprodutibilidade dos Testes , PublicaçõesRESUMO
The resazurin assay, also known as the Alamar Blue assay, stands as a cornerstone technique in cell biology, microbiology, and drug development. It assesses the viability of cells through the conversion of resazurin into highly fluorescent resorufin. The resulting fluorescence intensity provides a reliable estimate of viable cell numbers. Cytotoxicity assays, such as the resazurin-based method, play a crucial role in the screening of potential drug candidates and in the assessment of pharmaceutical and chemical toxicity. In recent years, inconsistencies have arisen in pharmacogenomic studies, often due to poorly optimized laboratory protocols. These inconsistencies hinder progress in understanding how substances affect cell health, leading to unreliable findings. Thus, the need for standardized and rigorously optimized protocols is evident to ensure consistent and accurate results in cytotoxicity studies. This manuscript describes a standardized procedure for optimizing resazurin-based viability assays to improve the reliability of cytotoxicity data. This optimization approach focuses on critical experimental parameters and data quality, aiming to achieve a level of measurement imprecision of less than 20%. In conclusion, to address the critical issues of reproducibility and reliability, protocol standardization, such as the one described in this manuscript, can greatly enhance the credibility of cytotoxicity studies, ultimately advancing drug safety assessments.
Assuntos
Sobrevivência Celular , Oxazinas , Xantenos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Bioensaio/métodos , Reprodutibilidade dos TestesRESUMO
Standardization is crucial when culturing cells including human embryonic stem cells (hESCs) which are valuable for therapy development and disease modeling. Inherent issues regarding reproducibility of protocols are problematic as they hinder translation to good manufacturing practice (GMP), thus reducing clinical efficacy and uptake. Pluripotent cultures require standardization to ensure that input material is consistent prior to differentiation, as inconsistency of input cells creates end-product variation. To improve protocols, developers first must understand the cells they are working with and their related culture dynamics. This innovative work highlights key conditions required for optimized and cost-effective bioprocesses compared to generic protocols typically implemented. This entailed investigating conditions affecting growth, metabolism, and phenotype dynamics to ensure cell quality is appropriate for use. Results revealed critical process parameters (CPPs) including feeding regime and seeding density impact critical quality attributes (CQAs) including specific metabolic rate (SMR) and specific growth rate (SGR). This implied that process understanding, and control is essential to maintain key cell characteristics, reduce process variation and retain CQAs. Examination of cell dynamics and CPPs permitted the formation of a defined protocol for culturing H9 hESCs. The authors recommend that H9 seeding densities of 20,000 cells/cm2, four-day cultures or three-day cultures following a recovery passage from cryopreservation and 100% medium exchange after 48 hours are optimal. These parameters gave ~SGR of 0.018 hour-1 ± 1.5x10-3 over three days and cell viabilities ≥95%±0.4, while producing cells which highly expressed pluripotent and proliferation markers, Oct3/4 (>99% positive) and Ki-67 (>99% positive).
Assuntos
Técnicas de Cultura de Células/métodos , Células-Tronco Embrionárias Humanas/citologia , Linhagem Celular , Proliferação de Células , Células Cultivadas , Humanos , Antígeno Ki-67/metabolismo , L-Lactato Desidrogenase/metabolismo , Ácido Láctico/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Fator de Transcrição PAX6/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismoRESUMO
To avoid loss of genetic information in environmental DNA (eDNA) field samples, the preservation of nucleic acids during field sampling is a critical step. In the development of standard operating procedures (SOPs) for eDNA-based compliance monitoring, the effect of different routinely used sediment preservations on biological community structures serving as bioindicators has gone untested. We compared eDNA metabarcoding results of marine bacterial communities from sample aliquots that were treated with a nucleic acid preservation solution (treated samples) and aliquots that were frozen without further treatment (non-treated samples). Sediment samples were obtained from coastal locations subjected to different stressors (aquaculture, urbanization, industry). DNA extraction efficiency, bacterial community profiles, and measures of alpha- and beta-diversity were highly congruent between treated and non-treated samples. As both preservation methods provide the same relevant information to environmental managers and regulators, we recommend the inclusion of both methods into SOPs for biomonitoring in marine coastal environments.
Assuntos
Monitoramento Biológico , Ecossistema , Biodiversidade , Código de Barras de DNA Taxonômico , Monitoramento Ambiental , GenômicaRESUMO
Down syndrome (DS), caused by trisomy of chromosome 21 (Hsa21), results in a spectrum of phenotypes including learning and memory deficits, motor dysfunction and social constrains. The regions on Hsa21 are conserved with their synteny on mouse chromosome 10, 16 and 17. To date, a wide range of mouse models has been developed to determine genotype-phenotype relationships and identity of the causative dosage-sensitive genes. However, the comparison of behavioral results is not obvious due to the lack of consistency in the genetics background, housing conditions and behavioral protocols used. There is a growing need to standardize some of the classical behavioral test, include automated behavioral phenotyping and sophisticated analysis techniques and move through ethologically inspired tests. Here we present an overview of the status of behavioral phenotyping of DS murine models and the limitations and possibilities to improve their characterization to address genotype-phenotype relationships for understanding the pathophysiology of DS.
Assuntos
Comportamento Animal , Modelos Animais de Doenças , Síndrome de Down/fisiopatologia , Endofenótipos , Atividade Motora/fisiologia , Comportamento Social , AnimaisRESUMO
This study aimed to investigate the influence of immersion duration and the type of immersion solution on the outcome of push-out bond strength (POBS) tests. Root canals of 120 straight single-rooted teeth were instrumented to a diameter of 1.5 mm and irrigated with 5 mL of 3% NaOCl. Four horizontal slices with a thickness of 1 mm were cut, representing the mid-portion of the root. The specimens (n = 480) were irrigated with 17% ethylenediaminetetraacetic acid(EDTA) for 60 seconds, then twice with distilled water (DW) for 30 s each. The canals were filled with either AH Plus (Dentsply Sirona, Konstanz, Germany) or BioRoot RCS (Septodont, St. Maur-des-Fossés, France) (n = 240). Separated into four groups per type of sealer (n = 60), the specimens were incubated at 37 °C covered with gauze moistened in DW or phosphate-buffered saline (PBS) for either one or eight weeks. Dislodgement resistance was measured and POBS was calculated. Statistical analysis was performed using the analysis of variance (ANOVA) test and the Student-Newman-Keuls test (p = 0.05). AH Plus showed higher POBS when stored in PBS compared to DW, irrespective of the incubation period (p < 0.05). BioRoot RCS displayed higher POBS when stored in DW compared to PBS after eight weeks of incubation (p < 0.05). No difference was found after one week of incubation (p > 0.05). Irrespective of the sealer or the immersion solution, POBS decreased from one week to eight weeks (p < 0.05). Mixed failure modes were found in all groups irrespective of sealer, immersion medium, or immersion period. POBS decreased after a longer incubation time in both immersion solutions. Duration of immersion and the type of immersion solution had a significant impact on the outcome of the POBS testing protocol.
RESUMO
With the advent of novel strategies to induce tolerance in autoimmune and autoimmune-like conditions, clinical trials of antigen-specific tolerizing immunotherapy have become a reality. Besides safety, it will be essential to gather mechanistic data on responding CD4+ T cells to assess the effects of various immunomodulatory approaches in early-phase trials. Peptide-MHC class II (pMHCII) multimers are an ideal tool for monitoring antigen-specific CD4+ T cell responses in unmanipulated cells directly ex vivo. Various protocols have been published but there are reagent and assay limitations across laboratories that could hinder their global application to immune monitoring. In this methodological analysis, we compare protocols and test available reagents to identify sources of variability and to determine the limitations of the tetramer binding assay. We describe a robust pMHCII flow cytometry-based assay to quantify and phenotype antigen-specific CD4+ T cells directly ex vivo from frozen peripheral blood mononuclear cell samples, which we suggest should be tested across various laboratories to standardize immune-monitoring results.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Citometria de Fluxo/métodos , Antígenos de Histocompatibilidade Classe II/imunologia , Monitorização Imunológica/métodos , Peptídeos/imunologia , Anticorpos Monoclonais/imunologia , Doenças Autoimunes/imunologia , Humanos , Tolerância Imunológica/imunologia , Terapia de ImunossupressãoRESUMO
In this study we examined 6080 data gathered by our research group during more than 20 years of research on the moss biomonitoring technique, in order to quantify the variability generated by different aspects of the protocol and to calculate the overall measurement uncertainty associated with the technique. The median variance of the concentrations of different pollutants measured in moss tissues attributed to the different methodological aspects was high, reaching values of 2851 (ng·g-1)2 for Cd (sample treatment), 35.1 (µg·g-1)2 for Cu (sample treatment), 861.7 (ng·g-1)2 and for Hg (material selection). These variances correspond to standard deviations that constitute 67, 126 and 59% the regional background levels of these elements in the study region. The overall measurement uncertainty associated with the worst experimental protocol (5 subsamples, refrigerated, washed, 5 × 5 m size of the sampling area and once a year sampling) was between 2 and 6 times higher than that associated with the optimal protocol (30 subsamples, dried, unwashed, 20 × 20 m size of the sampling area and once a week sampling), and between 1.5 and 7 times higher than that associated with the standardized protocol (30 subsamples and once a year sampling). The overall measurement uncertainty associated with the standardized protocol could generate variations of between 14 and 47% in the regional background levels of Cd, Cu, Hg, Pb and Zn in the study area and much higher levels of variation in polluted sampling sites. We demonstrated that although the overall measurement uncertainty of the technique is still high, it can be reduced by using already well defined aspects of the protocol. Further standardization of the protocol together with application of the information on the overall measurement uncertainty would improve the reliability and comparability of the results of different biomonitoring studies, thus extending use of the technique beyond the context of scientific research.
Assuntos
Poluentes Atmosféricos/análise , Poluição do Ar/estatística & dados numéricos , Briófitas/metabolismo , Monitoramento Ambiental/métodos , Metais Pesados/análise , Manejo de Espécimes , Incerteza , Poluentes Atmosféricos/metabolismo , Indústrias , Metais Pesados/metabolismo , Material Particulado/análise , Reprodutibilidade dos Testes , Espanha , Oligoelementos/análiseRESUMO
PURPOSE: Attempts by magnetic resonance (MR) manufacturers to help imaging centres improve patient throughput has led to the development of more automated acquisition. This software is capable of customizing individual scan alignment; potentially improving imaging efficiency and standardizing protocols. However, substantial investments are required to introduce such systems, potentially deterring their widespread application. This study assessed the implementation costs and reduction in examination durations for automated knee MR imaging (MRI) software. MATERIALS AND METHODS: Research activities were performed at a community-based academic centre on a 3-Tesla (3-T) system using Siemens' Day Optimizing Throughput (Dot) knee software. Examination acquisition times were extracted from the system before and after software implementation. Fiscal year 2012/13 finances were used to determine the average hourly cost of MRI utilization. Costs associated with automated software implementation were also calculated. Finally, the number of knee scans required to achieve a positive return on investment using the software was established. RESULTS AND DISCUSSION: The mean (standard deviation, sample size) pre- and post-Dot software scan times were 23.20 (4.18, n = 266) and 21.94 (4.51, n = 59) minutes, respectively, for a routine knee scan and 11.88 (1.60, n = 74) and 11.24 (1.51, n = 27) minutes, respectively, for a fast knee scan. The overall weighted average resulted in a 64-second time savings per automated knee examination. This negligible time savings would be extremely difficult to make use of clinically. Dot simplified 29 unique knee protocols to two, improving the consistency of knee examinations. Current Dot software is not compatible with all patients and therefore has limitations that are a concern among MR technologists. CONCLUSION: Adoption of automated knee systems could assist in standardizing protocols; however, the cost of implementation and difficulty in modifying patient scheduling to reflect the minimal time savings would make a financial return unlikely to occur at small- and medium-sized institutions.
RESUMO
The research in extracellular vesicles (EVs) has been rising during the last decade. However, there is no clear consensus on the most accurate protocol to isolate and analyze them. Besides, most of the current protocols are difficult to implement in a hospital setting due to being very time-consuming or to requirements of specific infrastructure. Thus, our aim is to compare five different protocols (comprising two different medium-speed differential centrifugation protocols; commercially polymeric precipitation - exoquick - acid precipitation; and ultracentrifugation) for blood and urine samples to determine the most suitable one for the isolation of EVs. Nanoparticle tracking analysis, flow cytometry, western blot (WB), electronic microscopy, and spectrophotometry were used to characterize basic aspects of EVs such as concentration, size distribution, cell-origin and transmembrane markers, and RNA concentration. The highest EV concentrations were obtained using the exoquick protocol, followed by both differential centrifugation protocols, while the ultracentrifugation and acid-precipitation protocols yielded considerably lower EV concentrations. The five protocols isolated EVs of similar characteristics regarding markers and RNA concentration; however, standard protocol recovered only small EVs. EV isolated with exoquick presented difficult to be analyzed with WB. The RNA concentrations obtained from urine-derived EVs were similar to those obtained from blood-derived ones, despite the urine EV concentration being 10-20 times lower. We consider that a medium-speed differential centrifugation could be suitable to be applied in a hospital setting as it requires the simplest infrastructure and recovers higher concentration of EV than standard protocol. A workflow from sampling to characterization of EVs is proposed.