Sample Handling Protocols Research Articles

Systematic evaluation supports the use of ELISA for quantification of calprotectin in equine feces, a first step toward noninvasive quantification of intestinal inflammation in horses.

To optimize and evaluate methods for the detection of the inflammatory biomarkers myeloperoxidase (MPO) and calprotectin (CP) in equine feces by ELISA. Healthy horses (n = 28) and horses with intestinal inflammation (n = 10). Feces were suspended in buffer to create fecal supernatant. Serum and fecal supernatant were analyzed using ELISA kits validated for the detection of MPO and CP in equine serum. Assay validation steps included intra- and interassay variability (coefficient of variation [CV]), dilution linearity, spike recovery, and sample type correlation. Variations in sample handling protocols (centrifugation speed, extraction buffer, and filtration) were evaluated. 17 paired fecal and serum samples were used for initial analysis (10 healthy horses, 7 colitis). Previously reported sample handling protocols resulted in detectable MPO and CP but poor CV, linearity, and spike recovery. There was a linear correlation between serum and fecal samples for CP but not MPO. There was a significant difference between the concentration and CV of alternative sample handling protocols for CP and MPO, with improved CV for CP (2.1% to 18.6%) but not MPO (14.4% to 53.4%). Processing fresh feces with a fecal extraction buffer and filtration of supernatant resulted in the best CV (0.5% to 3.8%) and recovery (45% to 64%) for CP. Detection of MPO was inconsistent regardless of method. There are few reliable diagnostic modalities for inflammation of the equine large colon. Findings support quantification of CP in equine feces using the described ELISA kit and protocol. With additional study to establish reference interval and clinical utility, the fecal inflammatory biomarker CP may allow for noninvasive quantification of intestinal inflammation in horses.

The appropriate sample-handling procedure for measuring the plasma β-amyloid level using a fully automated immunoassay

Plasma β-amyloid (Aβ) assays are a promising tool for Alzheimer’s disease diagnosis in clinical practice. To obtain reliable results, establishing an appropriate sample-handling procedure for each analytical platform is warranted. This study proposes an appropriate sample-handling procedure using HISCL analyzer by elucidating the individual/combined effects of pre-analytical parameters on plasma Aβ42/Aβ40 levels. We investigated the effects of various pre-analytical parameters, including storage times for whole blood, plasma, and freezing conditions, on plasma Aβ42/Aβ40 levels, and confirmed if these values met the acceptable criteria. Plasma Aβ42/Aβ40 levels were acceptable in all conditions. We determined our protocol by confirming that plasma Aβ42/Aβ40 levels remained acceptable when combining pre-analytical parameters. We established an appropriate sample-handling protocol that ensures reliable measurement of plasma Aβ42/Aβ40 levels using HISCL analyzer. We believe the Aβ assay, with our protocol, shows promise for aiding AD diagnosis in clinical settings.

Abstract 6186: Histopathology-derived molecular portrait of serous ovarian cancer

Abstract Background: The progression of ovarian cancer (OV) is often evaluated through clinical staging and molecular profiling of genomic alterations and subtypes, with the molecular profiling of tumors playing a crucial role in cancer management by informing therapeutic decisions. However, collecting molecular data is expensive, time-consuming, and can be sensitive to sample handling protocols. These factors prevent routine implementation at many care centers, thereby limiting benefit to patients. This work examines the viability of an image-centric method for inferring molecular endophenotypes and predicting overall survival in OV, leveraging commonly gathered histopathology scans. Methods: Embedding representations of digitized Hematoxylin and Eosin slides were produced using a self-supervised histology model. First, we assessed the ability of the embeddings to predict overall survival using Cox proportional hazards models by calculating the concordance index. Next, these embeddings served as input features to machine learning models trained to predict genomic patterns within tumors, including molecular subtypes, driver gene alterations, and mutational processes, leading to a more comprehensive analysis of the prognostic potential of our approach. Results: Histopathological embeddings were found to be more predictive of patient survival than traditional factors such as age, histological grade, and clinical stage. Embedding risk combined with clinicopathological risk had a higher predictive value than clinicopathological risk alone (0.578 vs 0.569, p = 0.025). Moreover, embedding risk alone was more predictive of survival (0.577 vs 0.569, p = 0.038). Risk scores derived from embeddings and all clinicopathological features were uncorrelated (R = 0.03), indicating that embeddings provide unique prognostic information. Motivated by these findings, we investigated the reasons behind the enhanced accuracy in survival prediction. We found that models trained on embeddings predicted previously defined molecular subtypes (TCGA, 2011), achieving area under the receiver operating characteristic curve (AUROC) scores as follows: Differentiated (0.86), Immunoreactive (0.85), Mesenchymal (0.9), and Proliferative (0.89). Similarly, our models identified crucial oncogenic events, including the loss of TP53 and CDKN2A, and instances of whole genome duplication (AUROCs of 0.85, 0.99 and 0.8). Conclusions: Taken together, this work showcases the potential utility of standard of care digitized histopathology scans for imputing genomic patterns and predicting overall survival in OV. Our results suggest that imputation could provide a fast and scalable solution for deploying patient screening biomarkers, thereby playing a pivotal role in shaping the future of precision medicine. Citation Format: Yajas Shah, Zachary R. McCaw, Anna Shcherbina, Christopher Probert. Histopathology-derived molecular portrait of serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6186.

Effects of certain pre-analytical factors on the performance of plasma phospho-tau217

IntroductionPre-analytical factors can cause substantial variability in the measurements of cerebrospinal fluid (CSF) and plasma biomarkers of Alzheimer’s disease (AD). However, their effects on the performance of one of the most promising plasma AD biomarkers, phosphorylated tau (p-tau)217, are not known.MethodsWe included 50 amyloid-β positive (Aβ+) and 50 Aβ− participants from the Swedish BioFINDER-1 study. Plasma and CSF p-tau217 were measured using an immunoassay developed by Lilly Research Laboratories. We examined the effect of four plasma handling conditions, i.e., (1) thawing at room temperature (RT) with no centrifugation, (2) thawing at RT followed by centrifugation, (3) thawing on ice with no centrifugation, and (4) thawing on ice followed by centrifugation. In addition, we also tested the effects of up to 3 freeze–thaw cycles on the associations of plasma p-tau217 with AD-related pathologies measured with CSF p-tau217 and CSF Aβ42/Aβ40.ResultsIn the whole cohort (combining Aβ+ and Aβ− participants), we found significant correlations between plasma p-tau217 and both CSF p-tau217 (Rrange, 0.614–0.717, p < 0.001) and CSF Aβ42/Aβ40 (Spearman Rrange, − 0.515 to − 0.652, p < 0.001) for each of the four tested conditions. Correlations between plasma and CSF p-tau217 were also significant for all conditions in the Aβ+ group (Rrange, 0.506–0.579, p < 0.001). However, in this Aβ+ subgroup, correlations with CSF Aβ42/Aβ40 were only significant for centrifuged samples (thawed at RT, R = − 0.394, p = 0.010; thawed on ice, R = − 0.406; p = 0.007). In Aβ− participants, correlations between plasma and CSF p-tau217 were again significant only for centrifuged samples (thawed at RT, R = 0.394, p = 0.007; thawed on ice, R = 0.334; p = 0.022), with no correlations seen between plasma p-tau217 and CSF Aβ42/Aβ40 for any of the conditions. While the accuracy of plasma p-tau217 to identify individuals with abnormal CSF Aβ42/Aβ40 or CSF p-tau217 status was high, the AUCs for samples thawed at RT and analyzed without centrifugation were numerically lower than the AUCs of other conditions (CSF Aβ42/Aβ40 = 0.845 vs 0.872–0.884; CSF p-tau217 = 0.866 vs 0.908–0.924, pdiff > 0.11). P-tau217 concentration was consistently higher in non-centrifuged samples than in centrifuged samples (p ≤ 0.021). There were no differences between samples freeze-thawed once, twice, or three times.ConclusionCentrifugation improved the performance of plasma p-tau217, but thawing temperatures and up to three freeze–thaw cycles did not have a significant impact. These results may inform the future development of standardized sample-handling protocols for AD biomarkers.

Optimized Pre-analytical Handling Protocol for Blood-Based Biomarkers of Alzheimer's Disease.

The therapeutic management of patients with Alzheimer's disease (AD) has been hindered by poor diagnostic accuracy. As such, there is an unmet clinical need for tools that can detect and diagnose the disease in its early stages. Compared with cerebrospinal fluid (CSF)-based biomarkers or positron emission tomography(PET), the use of reliable blood-based biomarkers could offer an accessible and minimally invasive method of streamlining diagnosis in the clinical setting. However, the influence of pre-analytical processing and sample handling parameters on the accurate measurement of protein biomarkers is well established, especially for AD CSF-based biomarkers. In this chapter, we provide recommendations for an optimal sample handling protocol for the analysis of blood-based biomarkers specifically for amyloid pathology in AD.

The Hong Kong genome project: building genome sequencing capacity and capability for advancing genomic science in Hong Kong

Aim: The Hong Kong Genome Project (HKGP) is the first large-scale genome sequencing (GS) project in the Hong Kong Special Administrative Region. The Hong Kong Genome Institute (HKGI) is entrusted with the task of implementing the HKGP. With the aim to sequence 45,000-50,000 genomes in five years, it is the project’s goal to provide participants with more precise diagnosis and personalised treatment, and to drive the application and integration of genomic medicine into routine clinical care. Methods: The HKGI Laboratory’s hardware and software components were customised to tailor to the needs of the project. Sample handling and storage protocol, DNA extraction, and PCR-free GS workflow were developed and optimised. Quality control indicators and metrics for assessing the quality of samples, sequencing libraries and sequencing data were established. Results: The Laboratory is designed to facilitate a unidirectional GS workflow to minimise the risk of contamination. The Sample Manager system handles laboratory data generated from the HKGP samples and biobank. The Laboratory handles and analyses approximately 350-500 samples per week, the majority of which are whole blood. During the first 24 months since the launch of the HKGP, 12,937 participants and their family members (6,680 genomes) have been recruited and sequenced. The sequencing capacity of the Laboratory has been further enhanced to include the latest technologies, such as long-read sequencing and multi-omics in order to meet the target of the HKGP. Conclusion: HKGI Laboratory established a robust GS workflow for the HKGP. The clinical utility of GS will bring precision medicine into routine clinical practice.

Proof of concept of using a membrane-sensing peptide for sEVs affinity-based isolation.

Introduction: One main limitation in biomarker studies using EVs is the lack of a suitable isolation method rendering high yield and purity samples in a quick and easily standardized procedure. Here we report an affinity isolation method with a membrane-sensing peptide (MSP) derived from bradykinin. Methods: We designed a protocol based on agarose beads carrying cation chelates to specifically bind to the 6His-tagged membrane-sensing peptide. This approach presents several advantages: 1) cation-carrying agaroses are widely used and standardized for His-tagged protein isolation, 2) the affinity protocol can be performed in small volumes, feasible and manageable for clinical routine and 3) elution with imidazole or EDTA allows a gentle and easy recovery without EV damage, facilitating subsequent characterization and functional analyses. Results: The optimized final procedure incubates 0.5mg of peptide for 10min with 10µL of Long-arm Cobalt agarose before an overnight incubation with concentrated cell conditioned medium. EV downstream analyses can be directly performed on the agarose beads adding lysis or nucleic-acid extraction buffers, or gently eluted with imidazole or EDTA, rendering a fully competent EV preparation. Discussion: This new isolation methodology is based on the recognition of general membrane characteristics independent of surface markers. It is thus unbiased and can be used in any species EV sample, even in samples from animal or plant species against which no suitable antibodies exist. Being an affinity method, the sample handling protocol is very simple, less time-consuming, does not require specialized equipment and can be easily introduced in a clinical automated routine. We demonstrated the high purity and yield of the method in comparison with other commercially available kits. This method can also be scale up or down, with the possibility of analyzing very low amounts of sample, and it is compatible with any downstream analyses thanks to the gentle elution procedure.

Pre-analytical sample handling standardization for reliable measurement of metabolites and lipids in LC-MS-based clinical research

The emerging disciplines of lipidomics and metabolomics show great potential for the discovery of diagnostic biomarkers, but appropriate pre-analytical sample-handling procedures are critical because several analytes are prone to ex vivo distortions during sample collection. To test how the intermediate storage temperature and storage period of plasma samples from K3EDTA whole-blood collection tubes affect analyte concentrations, we assessed samples from non-fasting healthy volunteers (n = 9) for a broad spectrum of metabolites, including lipids and lipid mediators, using a well-established LC-MS-based platform. We used a fold change-based approach as a relative measure of analyte stability to evaluate 489 analytes, employing a combination of targeted LC-MS/MS and LC-HRMS screening. The concentrations of many analytes were found to be reliable, often justifying less strict sample handling; however, certain analytes were unstable, supporting the need for meticulous processing. We make four data-driven recommendations for sample-handling protocols with varying degrees of stringency, based on the maximum number of analytes and the feasibility of routine clinical implementation. These protocols also enable the simple evaluation of biomarker candidates based on their analyte-specific vulnerability to ex vivo distortions. In summary, pre-analytical sample handling has a major effect on the suitability of certain metabolites as biomarkers, including several lipids and lipid mediators. Our sample-handling recommendations will increase the reliability and quality of samples when such metabolites are necessary for routine clinical diagnosis.

Impact of pre-analytical sample handling factors on plasma biomarkers of Alzheimer's disease.

An unmet need exists for reliable plasma biomarkers of amyloid pathology, in the clinical laboratory setting, to streamline diagnosis of Alzheimer's disease (AD). For routine clinical use, a biomarker must provide robust and reliable results under pre-analytical sample handling conditions. We investigated the impact of different pre-analytical sample handling procedures on the levels of seven plasma biomarkers in development for potential routine use in AD. Using (1) fresh (never frozen) and (2) previously frozen plasma, we evaluated the effects of (A) storage time and temperature, (B) freeze/thaw (F/T) cycles, (C) anticoagulants, (D) tube transfer, and (E) plastic tube types. Blood samples were prospectively collected from patients with cognitive impairment undergoing investigation in a memory clinic. β-amyloid 1-40 (Aβ40), β-amyloid 1-42 (Aβ42), apolipoprotein E4, glial fibrillary acidic protein, neurofilament light chain, phosphorylated-tau (phospho-tau) 181, and phospho-tau-217 were measured using Elecsys® plasma prototype immunoassays. Recovery signals for each plasma biomarker and sample handling parameter were calculated. For all plasma biomarkers measured, pre-analytical effects were comparable between fresh (never frozen) and previously frozen samples. All plasma biomarkers tested were stable for ≤24 h at 4°C when stored as whole blood and ethylenediaminetetraacetic acid (EDTA) plasma. Recovery signals were acceptable for up to five tube transfers, or two F/T cycles, and in both polypropylene and low-density polyethylene tubes. For all plasma biomarkers except Aβ42 and Aβ40, analyte levels were largely comparable between EDTA, lithium heparin, and sodium citrate tubes. Aβ42 and Aβ40 were most sensitive to pre-analytical handling, and the effects could only be partially compensated by the Aβ42/Aβ40 ratio. We provide recommendations for an optimal sample handling protocol for analysis of plasma biomarkers for amyloid pathology AD, to improve the reproducibility of future studies on plasma biomarkers assays and for potential use in routine clinical practice.

Improving selective channel occlusion of complex hydrocarbons and fatty acid methyl esters in urea crystals by using an expendable 3D-printed microfluidic device for sample preparation in untargeted petroleomics

In this study, we describe a proof-of-concept investigation of the potential and limitations of employing channel occlusion for sample preparation in untargeted analysis in petroleomics. A middle petroleum distillate composed of fatty acid methyl esters (FAME) and a complex mixture of linear, branched, and cyclic hydrocarbons were selected as the model samples for this investigation. A microfluidic device was engineered to overcome the limitations of channel occlusion, resulting in a quick and robust method for sample preparation. The 3D-printed device using fused deposition modelling (FDM) allowed the combination of a 13-h multi-step sample handling protocol into a 2-min single-step procedure, which is also automation-friendly. Such developments were also evaluated using the analytical eco-scale to guide the development of a green analytical method. The relative standard deviation decreased 2-fold with method miniaturization. The efficiency of n-alkane removal was extended from tridecane (n-C13) to heptadecane (n-C17), compared to original method (n-C16 to n-C17). The analytical performance of the method was investigated for untargeted analysis. The tool used to probe the intra- and inter-class variance was multi-way principal component analysis (MPCA). MPCA modelling revealed that both methods generated equivalent chemical information, highlighting the benefits of reliable and reproducible sample preparation methods, especially for untargeted analysis. Such awareness is critical to avoid the generation of misleading results in fields that heavily rely on untargeted analysis and fingerprinting, such as petroleomics.

Synoptic assessment of coastal total alkalinity through community science

Comprehensive sampling of the carbonate system in estuaries and coastal waters can be difficult and expensive because of the complex and heterogeneous nature of near-shore environments. We show that sample collection by community science programs is a viable strategy for expanding estuarine carbonate system monitoring and prioritizing regions for more targeted assessment. ‘Shell Day’ was a single-day regional water monitoring event coordinating coastal carbonate chemistry observations by 59 community science programs and seven research institutions in the northeastern United States, in which 410 total alkalinity (TA) samples from 86 stations were collected. Field replicates collected at both low and high tides had a mean standard deviation between replicates of 3.6 ± 0.3 µmol kg−1 (σ mean ± SE, n = 145) or 0.20 ± 0.02%. This level of precision demonstrates that with adequate protocols for sample collection, handling, storage, and analysis, community science programs are able to collect TA samples leading to high-quality analyses and data. Despite correlations between salinity, temperature, and TA observed at multiple spatial scales, empirical predictions of TA had relatively high root mean square error >48 µmol kg−1. Additionally, ten stations displayed tidal variability in TA that was not likely driven by low TA freshwater inputs. As such, TA cannot be predicted accurately from salinity using a single relationship across the northeastern US region, though predictions may be viable at more localized scales where consistent freshwater and seawater endmembers can be defined. There was a high degree of geographic heterogeneity in both mean and tidal variability in TA, and this single-day snapshot sampling identified three patterns driving variation in TA, with certain locations exhibiting increased risk of acidification. The success of Shell Day implies that similar community science based events could be conducted in other regions to not only expand understanding of the coastal carbonate system, but also provide a way to inventory monitoring assets, build partnerships with stakeholders, and expand education and outreach to a broader constituency.

Preparation, Scanning and Analysis of Duckweed Using X-Ray Computed Microtomography.

Quantification of anatomical and compositional features underpins both fundamental and applied studies of plant structure and function. Relatively few non-invasive techniques are available for aquatic plants. Traditional methods such as sectioning are low-throughput and provide 2-dimensional information. X-ray Computed Microtomography (μCT) offers a non-destructive method of three dimensional (3D) imaging in planta, but has not been widely used for aquatic species, due to the difficulties in sample preparation and handling. We present a novel sample handling protocol for aquatic plant material developed for μCT imaging, using duckweed plants and turions as exemplars, and compare the method against existing approaches. This technique allows for previously unseen 3D volume analysis of gaseous filled spaces, cell material, and sub-cellular features. The described embedding method, utilizing petrolatum gel for sample mounting, was shown to preserve sample quality during scanning, and to display sufficiently different X-ray attenuation to the plant material to be easily differentiated by image analysis pipelines. We present this technique as an improved method for anatomical structural analysis that provides novel cellular and developmental information.

Assessing the Preanalytical Variability of Plasma and Cerebrospinal Fluid Processing and Its Effects on Inflammation-Related Protein Biomarkers

Proteomics studies are important for the discovery of new biomarkers as clinical tools for diagnosis and disease monitoring. However, preanalytical variations caused by differences in sample handling protocol pose challenges for assessing biomarker reliability and comparability between studies. The purpose of this study was to examine the effects of delayed centrifuging on measured protein levels in plasma and cerebrospinal fluid (CSF). Blood from healthy individuals and patients with multiple sclerosis along with CSF from patients with suspected neurological disorders were left at room temperature for different periods (blood: 1, 24, 48, 72 h; CSF: 1 and 6 h) prior to centrifuging. Ninety-one inflammation-related proteins were analyzed using a proximity extension assay, a high-sensitivity multiplex immunoassay. Additional metabolic and neurology-related markers were also investigated in CSF. In summary, many proteins, particularly in plasma, had increased levels with longer delays in processing likely due in part to intracellular leakage. Levels of caspase 8, interleukin 8, interleukin 18, sirtuin 2, and sulfotransferase 1A1 increased 2-fold to 10-fold in plasma after 24 h at room temperature. Similarly, levels of cathepsin H, ectonucleoside triphosphate diphosphohydrolase 5, and WW domain containing E3 ubiquitin protein ligase 2 differentiated in CSF with <6 h delay in processing. However, the rate of change for many proteins was relatively consistent; therefore, we were able to characterize biomarkers for detecting sample handling variability. Our findings highlight the importance of timely and consistent sample collection and the need for increased awareness of protein susceptibility to sample handling bias. In addition, suggested biomarkers may be used in certain situations to detect and correct for preanalytical variation in future studies.

A blood-based metabolomics test to distinguish relapsing\u2013remitting and secondary progressive multiple sclerosis: addressing practical considerations for clinical application

The transition from relapsing–remitting multiple sclerosis (RRMS) to secondary progressive MS (SPMS) represents a huge clinical challenge. We previously demonstrated that serum metabolomics could distinguish RRMS from SPMS with high diagnostic accuracy. As differing sample-handling protocols can affect the blood metabolite profile, it is vital to understand which factors may influence the accuracy of this metabolomics-based test in a clinical setting. Herein, we aim to further validate the high accuracy of this metabolomics test and to determine if this is maintained in a ‘real-life’ clinical environment. Blood from 31 RRMS and 28 SPMS patients was subjected to different sample-handling protocols representing variations encountered in clinics. The effect of freeze–thaw cycles (0 or 1) and time to erythrocyte removal (30, 120, or 240 min) on the accuracy of the test was investigated. For test development, samples from the optimised protocol (30 min standing time, 0 freeze–thaw) were used, resulting in high diagnostic accuracy (mean ± SD, 91.0 ± 3.0%). This test remained able to discriminate RRMS and SPMS samples that had experienced additional freeze–thaw, and increased standing times of 120 and 240 min with accuracies ranging from 85.5 to 88.0%, because the top discriminatory metabolite biomarkers from the optimised protocol remained discriminatory between RRMS and SPMS despite these sample-handling variations. In conclusion, while strict sample-handling is essential for the development of metabolomics-based blood tests, the results confirmed that the RRMS vs. SPMS test is resistant to sample-handling variations and can distinguish these two MS stages in the clinics.

To composite or replicate: how sampling method and protocol differences alter collected stream invertebrates and associated bioassessment metrics.

Aquatic invertebrates are excellent indicators of ecosystem quality; however, choosing a sampling method can be difficult. Each method and associated protocol has advantages and disadvantages, and finding the approach that minimizes biases yet fulfills management objectives is crucial. To test the effects of both sampling methods and sample handling-i.e., to composite samples or leave them as replicates-we collected aquatic invertebrates from the Niobrara River at Agate Fossil Beds National Monument, Nebraska, using three methods and two sample handling protocols. We compared aquatic invertebrate assemblages collected with a Hester-Dendy multi-plate sampler, Hess sampler, and a D-frame dipnet. We calculated six common bioassessment metrics from composite (combined) and replicate (separate) samples. Hess samples contained the highest taxonomic richness (capturing 77% of all taxa observed) and dipnet samples the least (47%). Hester-Dendy samples had the greatest proportion of Ephemeroptera, and Ephemeroptera, Plecoptera, and Trichoptera (EPT). Dipnet samples had the lowest evenness values. In terms of sample handling, composite samples had inflated richness, diversity, and evenness compared with replicate samples, but bioassessment metrics calculated from proportions or averages (i.e., Hilsenhoff's Biotic Index and the proportion of EPT taxa) did not differ between them. The proportion of invertebrate groups from composite samples were not statistically different among sampling methods, but several groups differed between replicate samples collected by different methods. Ultimately, we recommend collecting replicate samples with a Hess sampler when the goal of the study is to detect ecosystem change, among locations or differences in variables of interest.

Assessment of Pre-Analytical Sample Handling Conditions for Comprehensive Liquid Biopsy Analysis

Liquid biopsies as a minimally invasive approach have the potential to revolutionize molecular diagnostics. Yet, although protocols for sample handling and the isolation of circulating tumor DNA (ctDNA) are numerous, comprehensive guidelines for diagnostics and research considering all aspects of real-life multicenter clinical studies are currently not available. These include limitations in sample volume, transport, and blood collection tubes. We tested the impact of commonly used (EDTA and heparin) and specialized blood collection tubes and storage conditions on the yield and purity of cell-free DNA for the application in down-stream analysis. Moreover, we evaluated the feasibility of a combined workflow for ctDNA and tumor cell genomic testing and parallel flow cytometric analysis of leukocytes. For genomic analyses, EDTA tubes showed good results if stored for a maximum of 4 hours at room temperature or for up to 24 hours when stored at 4°C. Spike-in experiments revealed that EDTA tubes in combination with density gradient centrifugation allowed the parallel isolation of ctDNA, leukocytes, and low amounts of tumor cells (0.1%) and their immunophenotyping by flow cytometry and down-stream genomic analysis by whole genome sequencing. In conclusion, adhering to time and temperature limits allows the use of routine EDTA blood samples for liquid biopsy analyses. We further provide a workflow enabling the parallel analysis of cell-free and cellular features for disease monitoring and for clonal evolution studies.

Microplot Design and Plant and Soil Sample Preparation for 15Nitrogen Analysis.

Manynitrogen fertilizer studies evaluate the overall effect of a treatment on end-of-season measurements such as grain yield or cumulative N losses. A stable isotope approach is necessary to follow and quantify the fate of fertilizer derived N (FDN) through the soil-crop system. The purpose of this paper is to describe a small-plot researchdesign utilizing non-confined 15N enriched microplots for multiple soil and plant sampling events over two growing seasons and provide sample collection, handling, and processing protocols for total 15N analysis. The methods were demonstrated using a replicated study from south-central Minnesota planted to corn (Zea mays L.). Each treatment consisted of six corn rows (76 cm row-spacing) 15.2 m long with a microplot (2.4 m by 3.8 m) embedded at one end. Fertilizer-grade urea was applied at 135 kg N∙ha-1 at planting, while the microplot received urea enriched to 5 atom % 15N. Soil and plant samples were taken several times throughout the growing season, taking care to minimize cross-contamination by using separate tools and physically separating unenriched and enriched samples during all procedures. Soil and plant samples were dried, ground to pass through a 2 mm screen, and then ground to a flour-like consistency using a roller jar mill. Tracer studies require additional planning, sample processing time and manual labor, and incur higher costs for 15N enriched materials and sample analysis than traditional N studies. However, using the mass balance approach, tracer studies with multiple in-season sampling events allow the researcher to estimate FDN distribution through the soil-crop system and estimate unaccounted-for FDN from the system.

Investigation of the relationship between ionised and total calcium in dogs with ionised hypercalcaemia.

To investigate the relationship between total and ionised calcium concentrations in dogs with ionised hypercalcaemia and to evaluate how albumin influences this relationship. Initially, a reference interval for ionised and total calcium was established using a large population of healthy adult dogs. Our teaching hospital clinical database was searched to identify adult dogs with ionised hypercalcaemia between 2012 and 2017, a time frame when the same sample handling and analysis protocols were in place as for the healthy reference interval population. The relationship between ionised and total calcium concentrations was then examined in the ionised hypercalcaemia population. Based on biochemical analysis of 351 healthy adult dogs, a reference interval of 1.18 to 1.53 mmol/L for ionised calcium and 2.24 to 2.85 mmol/L for total calcium was established. Using these reference intervals, 63 dogs with ionised hypercalcaemia were identified, of which 23 did not have total hypercalcaemia. Only seven of the 23 dogs with ionised hypercalcaemia and total calcium below the upper limit of the reference interval had hypoalbuminemia. The majority of dogs with ionised hypercalcemia and normal total calcium had a modest increase in ionised calcium. If relying on total calcium alone, more than one third of dogs with ionised hypercalcaemia will be classified as normocalcaemic and the majority of these dogs had normal serum albumin.

The impact of storage buffer, DNA extraction method, and polymerase on microbial analysis

Next-generation sequencing approaches used to characterize microbial communities are subject to technical caveats that can lead to major distortion of acquired data. Determining the optimal sample handling protocol is essential to minimize the bias for different sample types. Using a mock community composed of 22 bacterial strains of even concentration, we studied a combination of handling conditions to determine the optimal conditions for swab material. Examining a combination of effects simulates the reality of handling environmental samples and may thus provide a better foundation for the standardization of protocols. We found that the choice of storage buffer and extraction kit affects the detected bacterial composition, while different 16S rRNA amplification methods only had a minor effect. All bacterial genera present in the mock community were identified with minimal levels of contamination independent of the choice of sample processing. Despite this, the observed bacterial profile for all tested conditions were significantly different from the expected abundance. This highlights the need for proper validation and standardization for each sample type using a mock community and blank control samples, to assess the bias in the protocol and reduce variation across the datasets.

Improving Sample Collection, Sample Processing, and Laboratory Analyses for Corn Stalk Nitrate Test

Core Ideas Corn stalk nitrate test results can help corn growers fine‐tune N management. Adoption of corn stalk nitrate test use can be enhanced with more practical field sampling protocols. Laboratory processing of corn stalk nitrate test samples can be simplified for reduced analytical cost. The corn (Zea mays L.) stalk nitrate test (CSNT) is a site‐specific, after‐the‐fact, diagnostic tool for N management of corn. Although considered effective, widespread adoption of CSNT is limited due to somewhat impractical sample collection, handling, and laboratory sample processing protocols. Five studies were conducted to evaluate the impact of alternative sampling, sample handling, and laboratory processing protocols on CSNT outcomes. Study 1 evaluated the effect of stalk length and position on CSNT values. In Study 2 timing of sampling or growth stage was evaluated. In Study 3 several sampling patterns within a field (samples taken within a corn row vs. perpendicular or under an angle to rows) were analyzed. Study 4 tested if (a) quartering of stalks, (b) delay in mailing of stalks to the laboratory, and (c) temperature during storage (refrigerator, freezer, room temperature) affected CSNT‐N. Laboratory grinding, extraction, and determination procedures were compared in Study 5. The results suggest that sampling protocols can be adjusted to include shorter stalk samples, taken within 5 d after silage harvest. Direction of sampling within a field did not impact CSNT‐N, eliminating the need for more labor‐intensive sampling. Also, quartering of stalks and different temperature during storage did not change CSNT‐N. Laboratories can use 2‐mm grinding, extraction with distilled water, and a nitrate specific ion electrode or flow injection analysis. We conclude that adjustments in sampling protocols and sample processing can be made, possibly increasing farmer acceptance of the test, and reducing the cost of sampling, sample processing, and laboratory analyses.