Importance of Block Randomization When Designing Proteomics Experiments.

Randomization is used in experimental design to reduce the prevalence of unanticipated confounders. Complete randomization can however create imbalanced designs, for example, grouping all samples of the same condition in the same batch. Block randomization is an approach that can prevent severe imbalances in sample allocation with respect to both known and unknown confounders. This feature provides the reader with an introduction to blocking and randomization, and insights into how to effectively organize samples during experimental design, with special considerations with respect to proteomics.

Preanalytical Sample Handling Conditions and Their Effects on the Human Serum Metabolome in Epidemiologic Studies.

Many epidemiologic studies use metabolomics for discovery-based research. The degree to which sample handling may influence findings, however, is poorly understood. In 2016, serum samples from 13 volunteers from the US Department of Agriculture's Beltsville Human Nutrition Research Center were subjected to different clotting (30 minutes/120 minutes) and refrigeration (0 minutes/24 hours) conditions, as well as different numbers (0/1/4) and temperatures (ice/refrigerator/room temperature) of thaws. The median absolute percent difference (APD) between metabolite levels and correlations between levels across conditions were estimated for 628 metabolites. The potential for handling artifacts to induce false-positive associations was estimated using variable hypothetical scenarios in which 1%-100% of case samples had different handling than control samples. All handling conditions influenced metabolite levels. Across metabolites, the median APD when extending clotting time was 9.08%. When increasing the number of thaws from 0 to 4, the median APD was 10.05% for ice and 5.54% for room temperature. Metabolite levels were correlated highly across conditions (all r's ≥ 0.84), indicating that relative ranks were preserved. However, if handling varied even modestly by case status, our hypotheticals showed that results can be biased and can result in false-positive findings. Sample handling affects levels of metabolites, and special care should be taken to minimize effects. Shorter room-temperature thaws should be preferred over longer ice thaws, and handling should be meticulously matched by case status.

Reverse phase protein arrays in acute leukemia: investigative and methodological challenges.

INTRODUCTION: Acute leukemia results from a series of mutational events that alter cell growth and proliferation. Mutations result in protein changes that orchestrate growth alterations characteristic of leukemia. Proteomics is a methodology appropriate for study of protein changes found in leukemia. The high-throughput reverse phase protein array (RPPA) technology is particularly well-suited for the assessment of protein changes in samples derived from clinical trials. AREAS COVERED: This review discusses the technical, methodological, and analytical issues related to the successful development of acute leukemia RPPAs. EXPERT COMMENTARY: To obtain representative protein sample lysates, samples should be prepared from freshly collected blood or bone marrow material. Variables such as sample shipment, transit time, and holding temperature only have minimal effects on protein expression. CellSave preservation tubes are preferred for cells collected after exposure to chemotherapy, and incorporation of standardized guidelines for antibody validation is recommended. A more systematic biological approach to analyze protein expression is desired, searching for recurrent patterns of protein expression that allow classification of patients into risk groups, or groups of patients that may be treated similarly. Comparing RPPA protein analysis between cell lines and primary samples shows that cell lines are not representative of patient proteomic patterns.

The TELL automatic sample changer for macromolecular crystallography.

In this paper, the design and functionalities of the high-throughput TELL sample exchange system for macromolecular crystallography is presented. TELL was developed at the Paul Scherrer Institute with a focus on speed, storage capacity and reliability to serve the three macromolecular crystallography beamlines of the Swiss Light Source, as well as the SwissMX instrument at SwissFEL.

A Prospective Evaluation of Point-of-Care Measurements of Maternal Glucose for the Diagnosis of Gestational Diabetes Mellitus.

BACKGROUND: Point-of-care (POC) measurement of glucose is currently recommended only for the monitoring of gestational diabetes mellitus (GDM). This prospective observational study evaluated the use of POC measurements of maternal glucose to diagnose GDM in women being screened selectively with a 1-step 75 g oral glucose tolerance test (OGTT). METHODS: The strictest preanalytic and analytic international laboratory standards were applied to measure maternal plasma glucose at fasting and at 1 and 2 h post glucose load. The recent International Association of Diabetes and Pregnancy Study Groups diagnostic criteria were used. At the same time, maternal capillary glucose was measured. Because of differences in plasma and capillary glucose measurements, regression analysis of POC capillary glucose results vs laboratory plasma glucose results was conducted. The regression equations for plasma glucose were derived in a derivation cohort (n = 102). These equations were applied in the validation cohort (n = 100). Predicted and actual plasma glucose values were compared. RESULTS: Of the 202 women screened, 36.6% were nulliparous, 56.4% were obese, and 81.2% were Irish-born. Two thirds had a single risk factor for GDM, and a third had multiple risk factors. Based on the plasma measurements, 53.5% had GDM. As a predictor of GDM, the diagnostic accuracy of POC measurement was 83.0% (95% confidence interval, 74.2-89.8). CONCLUSIONS: In high-resource settings where measures to inhibit glycolysis are implemented, the use of POC measurements for the diagnosis of GDM is not justified based on this study. In low- and medium-resource settings, where measures to inhibit glycolysis are not achievable, regression analysis using POC measurements may be acceptable compared with plasma samples subject to glycolysis.

A proposed sample handling of ovine cotyledon for proteomic studies.

Ovine trophoblast is a suitable material for placental studies. However, a universal protocol for handling ruminant cotyledon samples has still not been reported. Considering the villous structure of ovine cotyledon, we suggest procedures to prepare cotyledons with limited inherent contamination, using semi-dry conditions to avoid freezing damage and sample errors. The cytosolic water-soluble proteins were physically extracted from the frozen cotyledons. High homogeneity was demonstrated between the replicates of both tissue and extract samples. Importantly, the chemical lysis of placental crude extracts was necessary for protein separation and immunoreaction. The integrity of stored tissues was histologically validated using a formalin-fixed paraffin-embedded technique. Using label-free proteomics, we detected 388 Ovis protein-groups in at least two of three biological replicates of either the tissue or extract. Although the water-soluble proteins were dominated by hemoglobin subunits, ten proteins were identified exclusively in all extract replicates. The physical extraction selectively reduced the membrane, extracellular matrix, and cytoskeleton proteins. The hydrolase enzymes, in the extract, hindered the identification of some specific proteins, such as histone H2A. In summary, the proposed workflow may guide further proteomic investigations of ovine cotyledon biology. Furthermore, our proteomic data have inferred some potential mechanisms of ovine trophoblast at parturition.

Practical Tips on Sample Handling for Hematology, Chemistry, and Cytology Testing for Equine Patients:: Getting More Bang for your Buck.

Clinical pathology results are only as good as the quality of samples and accompanying information submitted to the diagnostic laboratory. The frustration of nondiagnostic or equivocal test results can often be avoided by taking the time to follow sample handling and submission guidelines. This article discusses preanalytical errors that commonly affect the accuracy of hematology, chemistry, and cytology testing, and offers practical tips for preventing these errors and maximizing diagnostic yield.

Heparin and citrate additive carryover during blood collection.

Background Published evidence on the risk of additive carryover during phlebotomy remains elusive. We aimed to assess potential carryover of citrated and heparinized blood and the relative volume needed to bias clinical chemistry and coagulation tests. Methods We simulated standardized phlebotomies to quantify the risk of carryover of citrate and heparin additives in distilled water, using sodium and lithium as surrogates. We also investigated the effects of contamination of heparinized blood samples with increasing volumes of citrated blood and pure citrate on measurements of sodium, potassium, chloride, magnesium, total and ionized calcium and phosphate. Likewise, we studied the effects of contamination of citrated blood samples with increasing volumes of heparinized blood on heparin (anti-Xa) activity, lithium, activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT). We interpreted these results based on measurement deviations beyond analytical, biological and clinical significance. Results Standardized phlebotomy simulations revealed no significant differences in concentration of surrogate markers. Clinically significant alterations were observed after contamination of heparinized blood samples with volumes of citrated blood beyond 5-50 µL for ionized calcium and beyond 100-1000 µL for sodium, chloride and total calcium. Investigations of pure citrate carryover revealed similar results at somewhat lower volumes. Heparinized blood carryover showed clinically significant interference of coagulation testing at volumes beyond 5-100 µL. Conclusions Our results suggest that during a standardized phlebotomy, heparin or citrate contamination is highly unlikely. However, smaller volumes are sufficient to severely alter test results when deviating from phlebotomy guidelines.

Long-term Stability of Urinary Biomarkers of Acute Kidney Injury in Children.

BACKGROUND: Recent meta-analyses support the utility of urinary biomarkers for the diagnosis and prognosis of acute kidney injury. It is critical to establish optimal sample handling conditions for short-term processing and long-term urinary storage prior to widespread clinical deployment and meaningful use in prospective clinical trials. STUDY DESIGN: Prospective study. SETTING & PARTICIPANTS: 80 children (median age, 1.1 [IQR, 0.5-4.2] years) undergoing cardiac surgery with cardiopulmonary bypass at our center. 50% of patients had acute kidney injury (defined as ≥50% increase in serum creatinine from baseline). PREDICTORS: We tested the effect on biomarker concentrations of short-term urine storage in ambient, refrigerator, and freezer conditions. We also tested the effects of multiple freeze-thaw cycles, as well as prolonged storage for 5 years. OUTCOMES: Urine concentrations of neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule 1 (KIM-1), and interleukin 18 (IL-18). MEASUREMENTS: All biomarkers were measured using commercially available kits. RESULTS: All 3 biomarkers were stable in urine stored at 4°C for 24 hours, but showed significant degradation (5.6%-10.1% from baseline) when stored at 25°C. All 3 biomarkers showed only a small although significant decrease in concentration (0.77%-2.9% from baseline) after 3 freeze-thaw cycles. Similarly, all 3 biomarkers displayed only a small but significant decrease in concentration (0.84%-3.2%) after storage for 5 years. LIMITATIONS: Only the 3 most widely studied biomarkers were tested. Protease inhibitors were not evaluated. CONCLUSIONS: Short-term storage of urine samples for measurement of NGAL, KIM-1, and IL-18 may be performed at 4°C for up to 24 hours, but not at room temperature. These urinary biomarkers are stable at -80°C for up to 5 years of storage. Our results are reassuring for the deployment of these assays as biomarkers in clinical practice, as well as in prospective clinical studies requiring long-term urine storage.

Maintaining glucose integrity ex-vivo: Impact of preanalytical specimen handling.

BACKGROUND: Adopting the WHO protocol for glucose analysis is arguably impractical in the routine clinical setting. Deviations may develop due to a lack of understanding regarding the impact of glycolysis on the accuracy of results. AIM: We sought to assess the stability of glucose in two different blood collection tubes (BCT), BD Vacutainer® FX 'Fl-Ox' and Greiner Vacuette® FC-Mix 'FC-Mix' stored at room temperature (RT:18-22°C) and 4°C over 8.5 days. METHOD: Each participant provided venous whole blood collected into 51 BCTs; 'Fl-Ox' (n = 26) and 'FC-Mix' (n = 25). One Fl-Ox sample from each participant was handled according to the WHO recommended method. The remaining BCTs were stored at 4°C/RT prior to analyses at designated study timepoints. Glucose was measured using the hexokinase assay on the Cobas® 8000 platform. RESULTS: Participants (n = 8, Male = 2) were aged 24-56 years. Plasma glucose measured in FI-Ox BCTs according to the WHO sample-handling method had a median concentration of 5.73 mmol/L (Range: 5.39-10.37 mmol/L). Glucose decreased by greater than minimal difference (>0.26 mmol/L) in blood collected into Fl-Ox and stored @4°C/RT within 24 h of phlebotomy. FC-Mix BCT maintained glucose <0.26 mmol/L @4°C over a period of 8.5 days and up to 4 days @RT when compared to the WHO recommended method. CONCLUSION: Glucose in FC-Mix BCT stored @4°C demonstrated the best agreement with results determined using the WHO specifications. When FC-Mix tubes were stored @RT, glucose was stable for 4 days. These findings suggest that the FC-Mix BCT effectively inhibits glycolysis and should be introduced into routine clinical practice.

Analytical challenges related to the measurement of chlorothalonil in serum in the perspective of human biomonitoring studies.

Chlorothalonil (CTN) is a popular fungicide widely used in the world. However, its determination in serum samples is highly challenging, preventing a reliable investigation of human CTN internal exposure. We first investigated CTN's behaviour all along this analytical process on spiked serum samples. We used a radiolabelled 14C-CTN standard to monitor CTN in spiked serum samples and observed (1) a complete degradation of CTN in deproteinised serum samples after 4 h of contact; (2) a strong interaction between serum proteins and CTN by-products, with only 20 % of the radioactivity found to be extractable after 24 h of contact and (3) a slightly improved stability of CTN in serum following a first step of acidification or EDTA addition to samples. Using liquid chromatography coupled to high resolution mass spectrometry, 4-hydroxy-2,5,6-trichloroisophthalonitrile (HCTN) was identified as the major serum by-product of CTN. A protocol was developed to monitor both extractable CTN and HCTN from serum. This method was implemented on 36 human adult serum samples from the French "Esteban" Cohort. No free CTN was identified in these serum samples. Conversely, HCTN was detected in all samples at concentrations around 15 ± 2 ng mL-1, corresponding to the extractable fraction of CTN. Thus, HCTN may constitute a relevant biomarker of human internal exposure. Of note, the potential CTN contamination during blood collection could also be a source of HCTN detection in serum samples. Finally, blood sampling in EDTA tubes would seem more appropriate than in dry tubes for any future internal exposure studies on CTN.

Automation in Flow Cytometry.

Automation in clinical flow cytometry has the potential to revolutionize the field by improving processes and enhancing efficiency and accuracy. Integrating advanced robotics and artificial intelligence, these technologies can streamline sample preparation, data acquisition, and analysis. Automated sample handling reduces human error and increases throughput, allowing laboratories to handle larger volumes with consistent precision. Intelligent algorithms contribute to rapid data interpretation, aiding in the identification of cellular markers for disease diagnosis and monitoring. This automation not only accelerates turnaround times but also ensures reproducibility, making clinical flow cytometry a reliable tool in the realm of personalized medicine and diagnostics.

ALISTER - Application for lipid stability evaluation and research.

BACKGROUND AND AIMS: In lipidomic and metabolomic studies, pre-analytical pitfalls enhance the risk of misusing resources such as time and money, as samples that are analyzed may not yield accurate or reliable data due to poor sample handling. Guidance and pre-analytic know-how are necessary for translation of omics technologies into routine clinical testing. The present work aims to enable decision making regarding sample stability in every phase of lipidomics- and metabolomics-centered studies. MATERIALS AND METHODS: Data of multiple pre-analytic studies were aggregated into a database. Flexible approaches for evaluating these data were implemented in an RShiny-based web-application, tailored towards broad applicability in clinical and bioanalytic research. RESULTS: Our "Application for lipid stability evaluation & research" - ALISTER facilitates decision making on blood sample stability during lipidomic and metabolomic studies, such as biomarker research, analysis of biobank samples and clinical testing. The interactive tool gives sampling recommendations when planning sample collection or aids in the assessment of sample quality of experiments retrospectively. CONCLUSION: ALISTER is available for use under https://itmp.shinyapps.io/alister/. The application enables and simplifies data-driven decision making concerning pre-analytic blood sample handling and fits the needs of clinical investigations from multiple perspectives.

Adeno-associated virus analysis by size exclusion chromatography within 3 minutes using short bio-inert columns made with 3 µm particles operated at high flowrates.

Adeno-associated virus (AAV) analytical characterization is crucial to the well-defined and reproducible production of human gene therapies utilizing the AAV vector modality. The establishment of analytical methods based upon technology platforms currently widely used by bio-therapeutic manufacturers, namely HPLC, will assist efforts to produce high quality AAV reproducibly and decrease chemical manufacturing and control challenges in method portability and reliability. AAV analysis by size exclusion chromatography (SEC) is currently practiced with columns and mobile phase conditions traditional to SEC of proteins. Here, an improved method to measure multiple AVV critical quality attributes (CQA) rapidly by SEC is explored. The use of short columns made with small particles at high flow rates resulted in up to 80 % reduction in analysis time and 66 % in sample consumption while maintaining reliable quantitation of AAV aggregate or high molecular weight (HMW) content. These results were demonstrated across four different AAV serotypes. Furthermore, critical AAV sample handling learnings are shared.

Effects of Preanalytical Sample Collection and Handling on Comprehensive Metabolite Measurements in Human Urine Biospecimens.

Epidemiology studies evaluate associations between the metabolome and disease risk. Urine is a common biospecimen used for such studies due to its wide availability and non-invasive collection. Evaluating the robustness of urinary metabolomic profiles under varying preanalytical conditions is thus of interest. Here we evaluate the impact of sample handling conditions on urine metabolome profiles relative to the gold standard condition (no preservative, no refrigeration storage, single freeze thaw). Conditions tested included the use of borate or chlorhexidine preservatives, various storage and freeze/thaw cycles. We demonstrate that sample handling conditions impact metabolite levels, with borate showing the largest impact with 125 of 1,048 altered metabolites (adjusted P < 0.05). When simulating a case-control study with expected inconsistencies in sample handling, we predicted the occurrence of false positive altered metabolites to be low (< 11). Predicted false positives increased substantially (³63) when cases were simulated to undergo alternate handling. Finally, we demonstrate that sample handling impacts on the urinary metabolome were markedly smaller than those in serum. While changes in urine metabolites incurred by sample handling are generally small, we recommend implementing consistent handling conditions and evaluating robustness of metabolite measurements for those showing significant associations with disease outcomes.

Endocannabinoid analysis in GlucoEXACT plasma: Method validation and sample handling recommendations.

Endocannabinoids (ECs), such as anandamide and 2-arachidonyl glycerol (2-AG), contribute to the pathology of inflammatory, malignant, cardiovascular, metabolic and mental diseases. The reliability of quantitative analyses in biological fluids of ECs and endocannabinoid-like (EC-like) substances depends on pre-analytical conditions such as temperature and "time-to-centrifugation". Standardization of these parameters is critical for valid quantification and implementation in clinical research. In this study, we compared concentrations obtained with GlucoEXACT blood collection tubes versus K3EDTA tubes and employed the optimized procedure to assess ECs profiles in patients with inflammatory skin disease and healthy controls. A UHPLC-MS/MS method was validated for human plasma from GlucoEXACT blood collection tubes according to EMA and FDA guidelines, and pre-analytical conditions were systematically modified to assess analyte stability and optimize the procedures. The results showed significantly lower concentrations of ECs and EC-like substance concentrations with GlucoEXACT tubes compared with K3EDTA tubes, and GlucoEXACT extended the time window of stable concentrations. The strongest method-disagreement occurred for 1/2-AG suggesting that GlucoEXACT delayed ex vivo isomer rearrangement. Hence, GlucoExact tubes were superior in terms of stability and reliability. However, although absolute concentrations obtained with GlucoExact and K3EDTA differed, linear regression studies showed high agreement (except for 1/2-AG), and both methods showed similar EC profiles and similar disease-dependent pro-inflammatory patterns in dermatology patients. Hence, despite the obstacles in EC analyses, implementation of optimized pre-analytical blood collection and sample processing procedures provide reliable insight into peripheral ECs.

Fermentation Analytical Technology (FAT): Monitoring industrial E. coli fermentations using absolute quantitative 1H NMR spectroscopy.

BACKGROUND: To perform fast, reproducible, and absolute quantitative measurements in an automated manner has become of paramount importance when monitoring industrial processes, including fermentations. Due to its numerous advantages - including its inherent quantitative nature - Proton Nuclear Magnetic Resonance (1H NMR) spectroscopy provides an ideal tool for the time-resolved monitoring of fermentations. However, analytical conditions, including non-automated sample preparation and long relaxation times (T1) of some metabolites, can significantly lengthen the experimental time and make implementation in an industrial set up unfeasible. RESULTS: We present a high throughput method based on Standard Operating Procedures (SOPs) and 1H NMR, which lays the foundation for what we call Fermentation Analytical Technology (FAT). Our method was developed for the accurate absolute quantification of metabolites produced during Escherichia coli industrial fermentations. The method includes: (1) a stopped flow system for non-invasive sample collection followed by sample quenching, (2) automatic robot-assisted sample preparation, (3) fast 1H NMR measurements, (4) metabolites quantification using multivariate curve resolution (MCR), and (5) metabolites absolute quantitation using a novel correction factor (k) to compensate for the short recycle delay (D1) employed in the 1H NMR measurements. The quantification performance was tested using two sample types: buffer solutions of chemical standards and real fermentation samples. Five metabolites - glucose, acetate, alanine, phenylalanine and betaine - were quantified. Absolute quantitation ranged between 0.64 and 3.40 mM in pure buffer, and 0.71-7.76 mM in real samples. SIGNIFICANCE: The proposed method is generic and can be straight forward implemented to other types of fermentations, such as lactic acid, ethanol and acetic acid fermentations. It provides a high throughput automated solution for monitoring fermentation processes and for quality control through absolute quantification of key metabolites in fermentation broth. It can be easily implemented in an at-line industrial setting, facilitating the optimization of the manufacturing process towards higher yields and more efficient and sustainable use of resources.

Future of the Search for Life: Workshop Report.

The 2-week, virtual Future of the Search for Life science and engineering workshop brought together more than 100 scientists, engineers, and technologists in March and April 2022 to provide their expert opinion on the interconnections between life-detection science and technology. Participants identified the advances in measurement and sampling technologies they believed to be necessary to perform in situ searches for life elsewhere in our Solar System, 20 years or more in the future. Among suggested measurements for these searches, those pertaining to three potential indicators of life termed "dynamic disequilibrium," "catalysis," and "informational polymers" were identified as particularly promising avenues for further exploration. For these three indicators, small breakout groups of participants identified measurement needs and knowledge gaps, along with corresponding constraints on sample handling (acquisition and processing) approaches for a variety of environments on Enceladus, Europa, Mars, and Titan. Despite the diversity of these environments, sample processing approaches all tend to be more complex than those that have been implemented on missions or envisioned for mission concepts to date. The approaches considered by workshop breakout groups progress from nondestructive to destructive measurement techniques, and most involve the need for fluid (especially liquid) sample processing. Sample processing needs were identified as technology gaps. These gaps include technology and associated sampling strategies that allow the preservation of the thermal, mechanical, and chemical integrity of the samples upon acquisition; and to optimize the sample information obtained by operating suites of instruments on common samples. Crucially, the interplay between science-driven life-detection strategies and their technological implementation highlights the need for an unprecedented level of payload integration and extensive collaboration between scientists and engineers, starting from concept formulation through mission deployment of life-detection instruments and sample processing systems.

Submitting High-Quality Clinical Pathology Samples for Best Results.

The reliability of clinical pathology laboratory results is directly related to the sample quality submitted. As such, clinicians must submit the most representative and highest quality sample possible by acquiring, handling, preparing, and shipping samples with utmost care. Cytology and blood smear slides should be evaluated for sufficient densities of intact, well-spread, nucleated cells before submission. Poorly prepared samples may delay or negate results, incurring unnecessary costs for the client and practice. Additionally, all practices should have quality assurance programs that include monitoring of equipment to minimize reporting errors. Maximizing resources is the name of the game!.