Duration Of Sample Storage Research Articles

Investigating the association between hair progesterone, anxiety, sleep quality, and other determinants in South African females

BackgroundAnxiety symptoms and poor sleep quality are common in women. The role of sex hormones, particularly progesterone, in anxiety and sleep quality in women is understudied. Measurement of hair progesterone concentrations (HPC) is a promising method to investigate the effects of progesterone on anxiety symptoms and sleep quality in women. ObjectivesWe analysed sociodemographic, hair-related, and clinical factors associated with HPC and investigated the association between HPC and anxiety severity and sleep quality in a sample of 159 South African women (mean age: 46.5 years; range: 18–79 years). MethodsData were obtained from control participants from the SHARED ROOTS study. HPC were determined using an established liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. Unadjusted and adjusted multiple linear regression models were used to investigate whether sleep quality, measured with the Pittsburgh Sleep Quality Index (PSQI), and anxiety, measured with the Spielberger State-Trait Anxiety Inventory (STAI), were associated with HPC. Significant sociodemographic, hair-related, and clinical factors were adjusted for. ResultsHPC was significantly associated with age, duration of sample storage, hormonal treatment, postmenopausal status, and the number of different types of trauma exposures in adjusted models. Neither anxiety severity nor sleep quality was significantly associated with HPC. ConclusionsCertain demographic, hair related, and clinical factors were associated with HPC and need to be considered in future research using HPC. Although anxiety and sleep were not associated with HPC, greater trauma exposure was associated with higher HPC, suggesting an association between severe stress and hypothalamic-pituitary-gonadal (HPG) axis functioning.

Assessing the effect of sample storage time on viral detection using a rapid and cost-effective CTAB-based extraction method

BackgroundCassava leaf samples degrade quickly during storage and transportation from distant areas. Proper sampling and efficient, low-cost storage methods are critical for obtaining sufficient quality DNA and RNA for plant virus epidemiology and improving disease control understanding. This is useful when samples are collected from remote areas far from a laboratory or in developing countries where money and materials for virus diagnostics are scarce.ResultsThe effect of sample storage duration on nucleic acid (N.A.) quality on virus detection was investigated in this study. A simple, rapid, and cost-effective CTAB-based approach (M3) for single N.A. extraction was optimized and tested alongside two existing CTAB-based methods (M1 and M2) for N.A. extraction from fresh and herbarium cassava leaves stored for; 1, 8, 26, and 56 months. The amount and quality of DNA and RNA were determined using Nanodrop 2000 c U.V.–vis Spectrophotometer and agarose gel electrophoreses. The sample degradation rate was estimated using a simple mathematical model in Matlab computational software. The results show no significant difference in mean DNA concentration between M1 and M2 but a significant difference between M3 and the other two methods at p < 0.005. The mean DNA concentration extracted using M3 was higher for 1 and 8 months of leave storage. M3 and M2 produced high concentrations at 26 and 56 months of leave storage. Using a developed scale for quality score, M3 and M2 produced high-quality DNA from fresh samples. All methods produced poor-quality DNA and RNA at 8 and 26 months of leave storage and no visual bands at the age of 56 months. Statistically, there was a significant difference in the mean DNA quality between M1 and M2, but there was no significant difference between M3 and the other two methods at p < 0.005. However, Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV) were readily detected by RT-PCR from RNA isolated using M3. The quality of DNA declined per storage time at 0.0493 and 0.0521/month, while RNA was 0.0678 and 0.0744/month. Compared to the existing two methods, modified CTAB extracted enough high-quality N.A. in one-third the time of the existing two methods.ConclusionOur method provides cost-effective, quick, and simple processing of fresh and dry samples, which will quicken and guide the decision on when and what type of sample to process for plant disease management and surveillance actions.

Serum Metabolomic Markers of Protein-Rich Foods and Incident CKD: Results From the Atherosclerosis Risk in Communities Study

Rationale & ObjectiveWhile urine excretion of nitrogen estimates total protein intake, biomarkers of specific dietary protein sources have been sparsely studied. Using untargeted metabolomics, this study aimed to identify serum metabolomic markers of six protein-rich foods and to examine whether dietary protein-related metabolites are associated with incident chronic kidney disease (CKD). Study DesignProspective cohort study. Setting & Participants3,726 participants from the Atherosclerosis Risk in Communities (ARIC) study without CKD at baseline. ExposureDietary intake of six protein-rich foods (fish, nuts, legumes, red and processed meat, eggs, poultry), serum metabolites. OutcomesIncident CKD [eGFR <60 mL/min/1.73 m2 with ≥25% eGFR decline relative to visit 1, hospitalization or death related to CKD, or end-stage kidney disease. Analytical ApproachMultivariable linear regression models estimated cross-sectional associations between protein-rich foods and serum metabolites. C-statistics assessed the metabolites’ ability to improve discrimination of highest versus lower three quartiles of intake of protein-rich foods beyond covariates (demographics, clinical factors, health behaviors, and intake of nonprotein food groups). Cox regression models identified prospective associations between protein-related metabolites and incident chronic kidney disease (CKD). ResultsThirty significant associations were identified between protein-rich foods and serum metabolites (fish, n=8; nuts, n=5; legumes, n=0; red and processed meat, n=5; eggs, n=3; poultry, n=9). Metabolites collectively significantly improved discrimination of high intake of protein-rich foods compared to covariates alone (difference in C-statistics=0.033, 0.051, 0.003, 0.024, and 0.025 for fish, nuts, red and processed meat, eggs, and poultry-related metabolites, respectively; p<1.00 x 10-16 for all). Dietary intake of fish was positively associated with 1-docosahexaenoylglycerophosphocholine (22:6n3), which was inversely associated with incident CKD (HR 0.82, 95% CI 0.75-0.89, p=7.81×10-6). LimitationsResidual confounding and sample storage duration. ConclusionsWe identified candidate biomarkers of fish, nuts, red and processed meat, eggs, and poultry. A fish-related metabolite, 1-docosahexaenoylglycerophosphocholine (22:6n3), was associated with lower risk of CKD.

The effects of sample storage duration on tardigrade density and community composition in moss samples

In studies on micrometazoans, sample storage and processing methods are mostly decided based on sample quality (e.g., substrate type and moisture level), and the choice of methods may affect the reliability of the data. However, these methods are poorly studied and rarely reported in detail. Our aim was to determine the methodological compromise between efficiency and reliability required for large-scale quantitative meiofaunal ecological studies. Specifically, we tested whether storage duration (necessary for large number of samples) affects the density or community composition of tardigrades in moss samples. We focus on a largely unexplored limnoterrestrial ecosystem – boreal peatlands, where moss moisture levels are naturally variable across different microhabitats and moss species. We collected seven moss samples from a peatland in Central Finland, kept them in a refrigerator and extracted tardigrades using the Baermann wet funnel at 1, 24, 48 and 96 h post sampling. We found a significant decrease in tardigrade density (32 % on average), but no changes in community composition, after the first 24 h of storage. Based on these results, we recommend that samples collected from wet limnoterrestrial habitats should be processed within 24 h to ensure accuracy and comparability of large-scale quantitative data on tardigrade ecology.

The utilization of cryopreserved sperm in males with cancer.

e18791 Background: Ten percent of individuals diagnosed with cancer in the United States are under age 45. Fortunately, improvements in cancer therapy have enabled younger individuals to survive with cancer related deaths decreasing steadily over the past 20 years. The American Society for Clinical Oncology (ASCO) recognizes the importance of future family building for cancer survivors and states healthcare providers should discuss fertility preservation options prior to cancer treatment initiation. Despite these recommendations, the data regarding survivor utilization of cryopreservation sperm is limited. The aim of this study is to assess factors that impact the post treatment utilization of cryopreserved sperm. Methods: 721 patients who underwent sperm cryopreservation for cancer indication from 1/1/2010 – 10/1/2020 at a single institution were included in the retrospective chart analysis. Cancer diagnosis, patient age at time of sperm banking, sample storage duration, and sample disposition were collected. Sample disposition included utilization for pregnancy effort(s), request to discard and/or donate to research, and lost to follow up. Descriptive statistics were employed via Excel. Results: Fifty-two of 721 (7.21%, 95% CI 5.4-9.4%) patients utilized their sperm samples for fertility treatment, and 104 patients (14.42%) subsequently requested their samples be discarded. When stratified by age at time of sperm banking, men who banked between the ages of 36 and 40 years had the highest rate of sample utilization for pursuing fertility treatment (12/68, 17.65%, 95% CI 9.5-28.8%). Importantly, those that cryopreserved sperm and subsequently discarded the samples ranged from 8.97% (21 to 25 years) to 28.13% (41 to 45 years). When analyzed by diagnosis, patients who utilized their samples for fertility treatment had varying cancer diagnoses with percent utilization ranging from 4.11% (skin/connective tissue/musculoskeletal cancers) and 20.0% (lung cancer). Overall, 75.59% of the samples collected are unutilized (545/721, 95% CI of 72.3-78.7%). Only 3% were lost to follow up, defined as inability to contact patient after three attempts. Conclusions: Cryopreserved sperm is widely underutilized with only 7.21% of patients utilizing the samples for future conception. Men between ages 36 to 40 years at time of banking are most likely to utilize their samples and utilization varied by cancer diagnosis. This large cohort (with less than 5% lost to follow up) highlights the importance in identifying additional predictors of cryopreserved sperm utilization in cancer survivors. [Table: see text]

Parasite detection and quantification in avian blood is dependent on storage medium and duration.

Studies of parasites in wild animal populations often rely on molecular methods to both detect and quantify infections. However, method accuracy is likely to be influenced by the sampling approach taken prior to nucleic acid extraction. Avian Haemosporidia are studied primarily through the screening of host blood, and a range of storage mediums are available for the short- to long-term preservation of samples. Previous research has suggested that storage medium choice may impact the accuracy of PCR-based parasite detection, however, this relationship has never been explicitly tested and may be exacerbated by the duration of sample storage. These considerations could also be especially critical for sensitive molecular methods used to quantify infection (qPCR). To test the effect of storage medium and duration on Plasmodium detection and quantification, we split blood samples collected from wild birds across three medium types (filter paper, Queen's lysis buffer, and 96% ethanol) and carried out DNA extractions at five time points (1, 6, 12, 24, and 36 months post-sampling). First, we found variation in DNA yield obtained from blood samples dependent on theirstorage medium which had subsequent negative impacts on both detection and estimates of Plasmodium copy number. Second, we found that detection accuracy (incidence of true positives) was highest for filter-paper-stored samples (97%), while accuracy for ethanol and Queen's lysis buffer-stored samples was influenced by either storage duration or extraction yield, respectively. Lastly, longer storage durations were associated with decreased copy number estimates across all storage mediums; equating to a 58% reduction between the first- and third-year post-sampling for lysis-stored samples. These results raise questions regarding the utility of standardizing samples by dilution, while also illustrating the critical importance of considering storage approaches in studies of Haemosporidia comparing samples subjected to different storage regimes and/or stored for varying lengths of time.

The Effect of Storage at -20°C and -80°C on HbA1c Measurements in Whole Blood Samples Analysed Using Bio-Rad D-10

Introduction: Glycated haemoglobin (HbA1c) is a test commonly measured in many clinical laboratories to monitor and diagnose diabetes mellitus (DM). The laboratory in Hospital Tengku Ampuan Afzan (HTAA) receives HbA1c samples internally and from several other hospitals and clinics in the state of Pahang, at which, before transportation and analysis, the samples are often stored. This study determined the stability of HbA1c samples following different storage temperatures and duration for up to 30 days. Method: Whole blood samples for HbA1c analysis were collected from 222 healthy blood donors and type 2 DM (T2DM) patients. Each sample was prepared into four aliquots, which were then stored at temperatures -20°C and -80°C. HbA1c analyses were performed at baseline, days 15 and 30 using ion-exchange high-performance liquid chromatography (HPLC) assay technique on Bio-Rad D-10 analyser. HbA1c levels following sample storage were compared to the levels at baseline. Results: The baseline HbA1c (mean±SD) was 6.6±2.2%. At -20°C of storage, the HbA1c levels decreased with a mean difference of 0.1% and 0.3% on days 15 and 30, respectively. Storage at -80°C resulted in a mean difference of 0.1% on day 15 whilst no change was noted on day 30. The number of samples that showed clinically significant HbA1c change (defined as a change of &gt;0.5%) was less for the samples stored at -80°C (4.5%) as compared to -20°C (23%). Conclusion: HbA1c concentration is affected by temperature and duration of sample storage. In instances where prolonged storage of HbA1c samples is expected, storage at -80°C is preferable.

Second‐generation fully automated Elecsys cerebrospinal fluid immunoassays demonstrate high precision, reproducibility, and sample stability suitable for clinical use to aid Alzheimer’s disease diagnosis

AbstractBackgroundBeta‐amyloid(1–42) (Abeta42) and phosphorylated tau 181 (pTau) cerebrospinal fluid (CSF) biomarker analysis is recommended alongside cognitive evaluation to aid Alzheimer’s disease (AD) diagnosis. For clinical use, CSF biomarker assays should demonstrate high precision/reproducibility and sample stability. This study evaluated precision, reproducibility, and sample stability of second‐generation fully automated Elecsys® CSF immunoassays.MethodFrozen/pooled CSF and PreciControl samples were analyzed using Elecsys β‐Amyloid(1–42) CSF II/Phospho‐Tau (181P) CSF immunoassays on cobas e 601 (Roche Diagnostics International Ltd). pTau/Abeta42 ratios were calculated. Precision was evaluated at one site per Clinical and Laboratory Standards Institute (CLSI) EP02‐A3 (two runs/day in duplicate over 21 days; n=84). Reproducibility was determined per CLSI EP05‐A3 over five days using a single lot across three sites (site‐to‐site)/three lots at one site (lot‐to‐lot). Coefficients of variation (CVs)/standard deviations (SDs) for repeatability/intermediate precision/reproducibility were calculated using variance component analysis. Sample stability was determined from fresh CSF, collected per manufacturer's instructions. Baseline measurements were taken &lt;6 hours after lumbar puncture and follow‐up measurements (from stored aliquots) at defined intervals for ≤8 days at room temperature (RT; 25°C; n=13), ≤15 days at 6°C (n=14), and ≤13 weeks at ‐20°C (n=25). Storage stability was determined from percent recoveries/ordinary linear regression.ResultRepeatability CVs were &lt;2.4% and &lt;1.9% for Abeta42 and pTau immunoassays, respectively, and &lt;2.4% for pTau/Abeta42 ratio. Intermediate precision CVs were &lt;3.3% and &lt;3.7% for Abeta42 and pTau immunoassays, respectively, and &lt;3.9% for pTau/Abeta42 ratio. Site‐to‐site reproducibility CVs were &lt;5.1% and &lt;3.6% for Abeta42 and pTau immunoassays, respectively, and &lt;6.5% for pTau/Abeta42 ratio (Table 1). For the Abeta42 immunoassay, lot‐to‐lot reproducibility CVs were &lt;5.4%, except for one sample pool (mean concentration: 322 pg/mL; SD: 34.1 pg/mL; Table 2). Lot‐to‐lot reproducibility CVs were &lt;3.6% for the pTau immunoassay and &lt;6.0% for pTau/Abeta42 ratio. At maximum sample storage duration (8 days at RT/15 days at 2−8°C), median recoveries of 95%/98% were observed for Abeta42 and 97%/99% for pTau immunoassays. Stability assessment at ‐20°C is ongoing.ConclusionThe Elecsys immunoassays demonstrated high precision/reproducibility, supporting clinical use in AD diagnosis. Conservative recommendations for sample storage are 5 days at RT/14 days at 2−8°C.

Spatial transcriptomics tools allow for regional exploration of heterogeneous muscle pathology in the pre-clinical rabbit model of rotator cuff tear

BackgroundConditions affecting skeletal muscle, such as chronic rotator cuff tears, low back pain, dystrophies, and many others, often share changes in muscle phenotype: intramuscular adipose and fibrotic tissue increase while contractile tissue is lost. The underlying changes in cell populations and cell ratios observed with these phenotypic changes complicate the interpretation of tissue-level transcriptional data. Novel single-cell transcriptomics has limited capacity to address this problem because muscle fibers are too long to be engulfed in single-cell droplets and single nuclei transcriptomics are complicated by muscle fibers’ multinucleation. Therefore, the goal of this project was to evaluate the potential and challenges of a spatial transcriptomics technology to add dimensionality to transcriptional data in an attempt to better understand regional cellular activity in heterogeneous skeletal muscle tissue.MethodsThe 3′ Visium spatial transcriptomics technology was applied to muscle tissue of a rabbit model of rotator cuff tear. Healthy control and tissue collected at 2 and 16 weeks after tenotomy was utilized and freshly snap frozen tissue was compared with tissue stored for over 6 years to evaluate whether this technology is retrospectively useful in previously acquired tissues. Transcriptional information was overlayed with standard hematoxylin and eosin (H&E) stains of the exact same histological sections.ResultsSequencing saturation and number of genes detected was not affected by sample storage duration. Unbiased clustering matched the underlying tissue type-based on H&E assessment. Connective-tissue-rich areas presented with lower unique molecular identifier counts are compared with muscle fibers even though tissue permeabilization was standardized across the section. A qualitative analysis of resulting datasets revealed heterogeneous fiber degeneration–regeneration after tenotomy based on (neonatal) myosin heavy chain 8 detection and associated differentially expressed gene analysis.ConclusionsThis protocol can be used in skeletal muscle to explore spatial transcriptional patterns and confidently relate them to the underlying histology, even for tissues that have been stored for up to 6 years. Using this protocol, there is potential for novel transcriptional pathway discovery in longitudinal studies since the transcriptional information is unbiased by muscle composition and cell type changes.

Increasing the scope of the resazurin‐resorufin smart tracer system in hydrologic and biogeochemical sciences: The effects of storage duration and temperature on preservation

AbstractThe resazurin (raz)‐resorufin (rru) smart tracer system has led to significant advances in hydrologic and biogeochemical sciences but its application has been limited in scope and geography by the short duration that it has been assumed samples can be reliably stored before analysis. For the first time, we quantify the effects of sample storage duration and temperature on measured raz and rru concentrations in order to identify robust storage protocols. Raz/rru concentrations equivalent to those typically used in field and laboratory applications were prepared in three different mediums (deionized water, groundwater, and surface water). Samples were stored at 20°C, 5°C, and −20°C, representing average room, refrigerator, and freezer temperatures. Analysis of raz/rru concentration changes were conducted systematically during an 84‐day period, with higher frequency of analysis in the first 7 d. For samples stored at room temperature, changes of up to 22.6% were observed over the initial 24 h. In contrast, for samples stored in the refrigerator and freezer, changes of up to 8.4% and 6.9% were observed in the same period, respectively. Freezing samples provided the best preservation, yielding a maximum of ~ 10% change after 14 d compared to a maximum of ~ 30% change when cooled. It is generally recommended that raz/rru samples be stored cooled for up to 48 h and frozen for up to 14 d. This offers exciting opportunities to broaden the application of the raz‐rru system to undertake measurements in wider geographical (remote) locations and at increased sampling frequencies.

Pre-analytical factors affect the accurate measurement of testosterone concentrations in plasma and serum of goats.

Several pre-analytical factors may influence the accurate measurements of testosterone (T) and therefore, these factors must be a significant concern. This study aimed to examine the effects of 1) time of sample collection, 2) delay to centrifugation, 3) sample matrix types, and 4) device and duration of sample storage on the T concentrations. Blood samples were collected from 34 bucks of Kacang goats. For testing the effect of collection time, 12 pairs of morning and afternoon samples were collected. For testing the effect of delayed centrifugation, 24 samples were subjected to treatments: (i) centrifuged ⟨1 hour after collection (control group), (ii) centrifuged 6, 12, and 24 hours after collection (test groups). For testing the different sample matrix types, 10 samples were processed as serum and plasma. For testing the effect of sample storage device and duration, 60 samples were subjected to treatments: i) frozen at -20OC (control group), ii) stored in a cooler box, a styrofoam box, and a thermos-flask for two, four, and six days (test groups). T concentrations were measured using a validated testosterone ELISA kit. Concentrations of plasma testosterone (pT) from morning samples were significantly higher compared to afternoon samples (p⟨0.05). Delayed centrifugation for up to 24 h decreased significantly on pT concentrations (p⟨0.05). The concentrations of T from serum and plasma did not differ and showed a strong correlation (r=0.981). Storage device and duration affected the T concentrations compared to frozen samples (p⟨0.05) which T concentrations were stable for up to 4 days in a styrofoam box and a thermos-flask and up to 6 days in a cooler box. In conclusion, the measurement accuracy and stability of T concentrations in goats are affected by collection time, delay to centrifugation, and device and duration of storage.

Storage Duration Affects the Quantification of Oxidative Stress Markers in the Gastrocnemius, Heart, and Brain of Mice Submitted to a Maximum Exercise.

This study investigated the effect of sample storage duration on the quantification of oxidative stress markers in the gastrocnemius, heart, and brain of mice submitted to a maximum swimming exercise. Thiobarbituric acid reactive substances (TBARSs), protein carbonyl derivatives, total antioxidant capacity (TAC), and the activity of superoxide dismutase (SOD) and catalase (CAT) were quantified in fresh tissues and in samples stored at -80°C for 1, 3, or 6 months, from exercised (n = 13) and nonexercised mice (n = 13). Except for protein carbonyl derivatives in the heart, the exercise resulted in the modification of all markers in all fresh-evaluated samples (p < 0.001). The storage duration did not modify the effect of exercise on protein carbonyl derivatives and TAC. TBARS was stable for 3 months in the gastrocnemius and for 1 month in frozen heart and brain. Accordingly, the exercise effect on TBARS levels observed in fresh samples was absent in the gastrocnemius frozen for 6 months (p = 0.98) and in the heart and brain frozen for 3 months (p = 0.07 and 0.28, respectively) or more (p = 0.21 for heart and p > 0.99 for brain). In addition, CAT and SOD activities were reduced by storage duration in all tissues evaluated (p < 0.05). Our findings show that sample storage duration alters the quantification of oxidative stress markers in mice submitted to maximum exercise, and its effect is tissue and marker dependent. Some recommendations to achieve more accurate and reproducible data in the exercise physiology and oxidative stress markers field are presented.

Effect of initial moisture content and sample storage duration on compost stability using the ORG0020 dynamic respiration test

Biological tests are widely used to assess composting process status and finished material stability. Although compost stability is known to be influenced by moisture content (MC) and storage duration, there is a lack of data supporting boundary limits for standardised testing. Using the ORG0020 dynamic respiration test we assessed the stability of materials from different commercial composting sites processing only green waste or mixed green and food waste. Samples were tested at three different MC following adjustment with the ‘fist’ test within the range 40–60%. The results showed manipulation of MC within this range could have significant impact on measured stability for some but not all samples. Two samples reported significantly higher activity when MC was manipulated from ~50% to ~60%. For storage duration, samples showed significant decrease in measured activity over several weeks of cold storage. However, there was no significant difference in stability for samples tested up to nine days from receipt. The results of this research will support decisions relating to the boundary limits for moisture content and storage time for the ORG0020 test. The results will also provide insight to the wider range of biological tests used to assess compost stability.

Direct comparison study between droplet digital PCR and a combination of allele-specific PCR, asymmetric rapid PCR and melting curve analysis for the detection of BRAF V600E mutation in plasma from melanoma patients.

Background In metastatic melanoma, 40%-50% of patients harbor a BRAF V600E mutation and are thereby eligible to receive a combined BRAF/MEK inhibitor therapy. Compared to standard-of-care tissue-based genetic testing, analysis of circulating tumor DNA (ctDNA) from blood enables a comprehensive assessment of tumor mutational status in real-time and can be used for monitoring response to therapy. The aim of our study was to directly compare the performance of two highly sensitive methodologies, droplet digital PCR (ddPCR) and a combination of ARMS/asymmetric-rapid PCR/melting curve analysis, for the detection of BRAF V600E in plasma from melanoma patients. Methods Cell-free DNA (cfDNA) was isolated from 120 plasma samples of stage I to IV melanoma patients. Identical plasma-cfDNA samples were subjected to BRAF V600E mutational analysis using in parallel, ddPCR and the combination of ARMS/asymmetric-rapid PCR/melting curve analysis. Results BRAF V600E mutation was detected in 9/117 (7.7%) ctDNA samples by ddPCR and in 22/117 (18.8%) ctDNA samples by the combination of ARMS/asymmetric- rapid PCR/melting curve analysis. The concordance between these two methodologies was 85.5% (100/117). The comparison of plasma-ctDNA analysis using ddPCR and tissue testing revealed an overall agreement of 79.4% (27/34), while the corresponding agreement using the combination of ARMS/asymmetric-rapid PCR/melting curve analysis was 73.5% (25/34). Moreover, comparing the detection of BRAF-mutant ctDNA with the clinics, overall agreement of 87.2% (48/55) for ddPCR and 79.2% (42/53) was demonstrated. Remarkably, the duration of sample storage was negatively correlated with correctness of genotyping results highlighting the importance of pre-analytical factors. Conclusions Our direct comparison study has shown a high level of concordance between ddPCR and the combination of ARMS/asymmetric-rapid PCR/melting curve analysis for the detection of BRAF V600E mutations in plasma.

Microparticle Responses to Aerobic Exercise and Meal Consumption in Healthy Men.

Microparticles (MP) are shed extracellular vesicles that express the prothrombotic tissue factor (TF). Aerobic exercise may reduce MP count and TF expression. This study investigated the impact of acute running or rest followed by standardized meal consumption on MP phenotypes and TF expression. Fifteen males (age, 22.9 ± 3.3 yr; body mass, 81.9 ± 11.4 kg; V˙O2max, 54.9 ± 6.5 mL·kg·min; mean ± SD) completed 1 h of running (70% V˙O2max) or rest at 9:00 AM and consumed a standardized meal (1170 kcal, 43% CHO, 17% PRO, 40% fat) at 10:45 AM. Venous blood samples were taken at 9:00 AM, 10:00 AM, and 11:30 AM. The MP concentration, diameter, phenotypes, and TF expression were assessed using nanoparticle tracking analysis and flow cytometry. Nanoparticle tracking analysis identified no changes in MP concentration or diameter in response to time or trial. Flow cytometry revealed total MP count increased from 9:00 AM to 10:00 AM (1.62 ± 2.28 to 1.74 ± 2.61 × 10 L, P = 0.016, effect size (η) = 0.105), but was unaffected by trial. TF platelet-derived MP % reduced from 9:00 AM to 10:00 AM (44.0% ± 21.2% to 21.5% ± 9.3%, P = 0.001, η = 0.582) after exercise only (control, 36.8% ± 18.2% to 34.9% ± 11.9%; P = 0.972). TF neutrophil-derived MP percentage reduced from 9:00 AM to 11:30 AM (42.3% ± 17.2% to 25.1% ± 14.9%; P = 0.048, η = 0.801) in the exercise trial only (control, 28.5% ± 15.7% to 32.2% ± 9.6%; P = 0.508). Running induced a significant reduction in %TF platelet and neutrophil MP, suggesting a transient reduction in cardiovascular risk via reduced TF-stimulated thrombosis. This requires further investigation over longer periods in cardiovascular disease populations.

A comparative study of different laboratory storage conditions for enhanced DNA analysis of crime scene soil-blood mixed sample

Crime scene investigation is an important step in the entire criminal investigation process because this is where evidence is gathered. Blood from the perpetrator or victim of a crime can be left at crime scenes or transferred to other materials such as clothing, knives and guns. Most often, this body fluid is contaminated with soil at outdoor crime scenes but this might be the only or the most important evidence in solving a crime. This work aimed at identifying the most appropriate method of storing crime scene soil-blood mixed sample prior to analysis at the laboratory. Human blood was mixed with soil and stored at three different storage conditions (i.e., Room temperature/25°C, 4°C and −20°C). Samples stored at room temperature saw significant reduction in DNA concentration as storage time increased (P=0.001). Samples stored at 4°C saw a drastic decrease in DNA concentration just after two weeks of storage. By the eighth week of storage at 4°C, there was no detectable DNA (P=0.000). Samples stored at −20°C recorded no specific pattern in decrease or increase in DNA concentration for the entire 12week storage (P=0.324). There were full STR Profiles generated for room temperature stored samples and −20°C stored samples throughout the study. There were full, partial and null Profiles generated for 4°C stored samples depending on the sample storage duration. In conclusion, −20°C was identified as the best storage condition for soil-blood mixed sample followed by room temperature and 4°C, respectively.

Metabolic profiles derived from residual blood spot samples: A longitudinal analysis

Background: Secondary use of newborn screening dried blood spot samples include use for biomedical or epidemiological research. However, the effects of storage conditions on archival samples requires further examination. The objective of this study was to determine the utility of residual newborn samples for deriving reliable metabolic gestational age estimates. Methods: Residual newborn dried blood spot samples that had been stored for 2-, 4-, 6-, or 12-months in temperature controlled (21°C) conditions were re-analyzed for the full panel of newborn screening analytes offered by a provincial newborn screening lab in Ottawa, Canada. Data from re-analyzed samples were compared to corresponding baseline newborn screening values for absolute agreement, and Pearson and intraclass correlation. Performance of a gestational age estimation algorithm originally developed from baseline newborn screening values was then validated on data derived from stored samples. Results: A total of 307 samples were used for this study. 17-hydroxyprogesterone and newborn hemoglobin profiles measured by immunoassay and high-performance liquid chromatography, respectively, were among the most stable markers across all time points of analysis. Acylcarnitines exhibited the greatest degree of variation in stability upon repeat measurement. The largest shifts in newborn analyte profiles and the poorest performance of metabolic gestational age algorithms were observed when samples were analyzed 12-months after sample collection. Conclusions: Duration of sample storage, independent of temperature and humidity, affects newborn screening profiles and gestational age estimates derived from metabolic gestational dating algorithms. When considering use of dried blood spot samples either for clinical or research purposes, care should be taken when interpreting data stemming from secondary use.

Metabolic profiles derived from residual blood spot samples: A longitudinal analysis.

Background: Secondary use of newborn screening dried blood spot samples include use for biomedical or epidemiological research. However, the effects of storage conditions on archival samples requires further examination. The objective of this study was to determine the utility of residual newborn samples for deriving reliable metabolic gestational age estimates. Methods: Residual newborn dried blood spot samples that had been stored for 2-, 4-, 6-, or 12-months in temperature controlled (21°C) conditions were re-analyzed for the full panel of newborn screening analytes offered by a provincial newborn screening lab in Ottawa, Canada. Data from re-analyzed samples were compared to corresponding baseline newborn screening values for absolute agreement, and Pearson and intraclass correlation. Performance of a gestational age estimation algorithm originally developed from baseline newborn screening values was then validated on data derived from stored samples. Results: A total of 307 samples were used for this study. 17-hydroxyprogesterone and newborn hemoglobin profiles measured by immunoassay and high-performance liquid chromatography, respectively, were among the most stable markers across all time points of analysis. Acylcarnitines exhibited the greatest degree of variation in stability upon repeat measurement. The largest shifts in newborn analyte profiles and the poorest performance of metabolic gestational age algorithms were observed when samples were analyzed 12-months after sample collection. Conclusions: Duration of sample storage, independent of temperature and humidity, affects newborn screening profiles and gestational age estimates derived from metabolic gestational dating algorithms. When considering use of dried blood spot samples either for clinical or research purposes, care should be taken when interpreting data stemming from secondary use.

Effects of preservation method on canine (Canis lupus familiaris) fecal microbiota.

Studies involving gut microbiome analysis play an increasing role in the evaluation of health and disease in humans and animals alike. Fecal sampling methods for DNA preservation in laboratory, clinical, and field settings can greatly influence inferences of microbial composition and diversity, but are often inconsistent and under-investigated between studies. Many laboratories have utilized either temperature control or preservation buffers for optimization of DNA preservation, but few studies have evaluated the effects of combining both methods to preserve fecal microbiota. To determine the optimal method for fecal DNA preservation, we collected fecal samples from one canine donor and stored aliquots in RNAlater, 70% ethanol, 50:50 glycerol:PBS, or without buffer at 25 °C, 4 °C, and −80 °C. Fecal DNA was extracted, quantified, and 16S rRNA gene analysis performed on Days 0, 7, 14, and 56 to evaluate changes in DNA concentration, purity, and bacterial diversity and composition over time. We detected overall effects on bacterial community of storage buffer (F-value = 6.87, DF = 3, P < 0.001), storage temperature (F-value=1.77, DF = 3, P = 0.037), and duration of sample storage (F-value = 3.68, DF = 3, P < 0.001). Changes in bacterial composition were observed in samples stored in −80 °C without buffer, a commonly used method for fecal DNA storage, suggesting that simply freezing samples may be suboptimal for bacterial analysis. Fecal preservation with 70% ethanol and RNAlater closely resembled that of fresh samples, though RNAlater yielded significantly lower DNA concentrations (DF = 8.57, P < 0.001). Although bacterial composition varied with temperature and buffer storage, 70% ethanol was the best method for preserving bacterial DNA in canine feces, yielding the highest DNA concentration and minimal changes in bacterial diversity and composition. The differences observed between samples highlight the need to consider optimized post-collection methods in microbiome research.

A selenium species in cerebrospinal fluid predicts conversion to Alzheimer\u2019s dementia in persons with mild cognitive impairment

BackgroundLittle is known about factors influencing progression from mild cognitive impairment to Alzheimer’s dementia. A potential role of environmental chemicals and specifically of selenium, a trace element of nutritional and toxicological relevance, has been suggested. Epidemiologic studies of selenium are lacking, however, with the exception of a recent randomized trial based on an organic selenium form.MethodsWe determined concentrations of selenium species in cerebrospinal fluid sampled at diagnosis in 56 participants with mild cognitive impairment of nonvascular origin. We then investigated the relation of these concentrations to subsequent conversion from mild cognitive impairment to Alzheimer’s dementia.ResultsTwenty-one out of the 56 subjects developed Alzheimer’s dementia during a median follow-up of 42 months; four subjects developed frontotemporal dementia and two patients Lewy body dementia. In a Cox proportional hazards model adjusting for age, sex, duration of sample storage, and education, an inorganic selenium form, selenate, showed a strong association with Alzheimer’s dementia risk, with an adjusted hazard ratio of 3.1 (95% confidence interval 1.0–9.5) in subjects having a cerebrospinal fluid content above the median level, compared with those with lower concentration. The hazard ratio of Alzheimer’s dementia showed little departure from unity for all other inorganic and organic selenium species. These associations were similar in analyses that measured exposure on a continuous scale, and also after excluding individuals who converted to Alzheimer’s dementia at the beginning of the follow-up.ConclusionsThese results indicate that higher amounts of a potentially toxic inorganic selenium form in cerebrospinal fluid may predict conversion from mild cognitive impairment to Alzheimer’s dementia.