Introduction of BD Vacutainer® Barricor™ tubes in clinical biobanking and application of amino acid and cytokine quality indicators to Barricor plasma.

OBJECTIVES: The use of BD Vacutainer® Barricor™ tubes (BAR) can reduce turnaround time (TAT) and improve separation of plasma from cellular components using a specific mechanical separator. Concentrations of amino acids (AAs) and cytokines, known to be labile during pre-analytical time delays, were compared in heparin (BAR, BD Heparin standard tube [PST]), EDTA and serum gel tubes (SER) to validate previously identified quality indicators (QIs) in BAR. METHODS: Samples of healthy individuals (n=10) were collected in heparin, EDTA and SER tubes and exposed to varying pre- and post-centrifugation delays at room temperature (RT). Cytokines (interleukin [IL]-8, IL-16 and sCD40L) were analyzed by enzyme-linked immunosorbent assay (ELISA) and AAs were characterized by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). RESULTS: All QIs, AAs/AA ratio and cytokines increased during prolonged blood storage in heparin plasma (PST, BAR) and SER tubes. Comparison of 53 h/1 h pre-centrifugation delay resulted in an increase in taurine (Tau) and glutamic acid (Glu) concentrations by more than three times, soluble CD40L increased by 13.6, 9.2 and 4.3 fold in PST, BAR-CTRL and BAR-FAST, and IL-8 increased even more by 112.8 (PST), 266.1 (BAR-CTRL), 268.1 (BAR-FAST) and 70.0 (SER) fold, respectively. Overall, compared to prolonged blood storage, effects of post-centrifugation delays were less pronounced in all tested materials. CONCLUSIONS: BAR tubes are compatible with the use of several established QIs and can therefore be used in clinical biobanking to reduce pre-analytical TAT without compromising QIs and thus pre-analytical sample quality analysis.

Proficiency Testing to Assess Technical Performance for CTC-Processing and Detection Methods in CANCER-ID.

BACKGROUND: Multiple technologies are available for detection of circulating tumor cells (CTCs), but standards to evaluate their technical performance are still lacking. This limits the applicability of CTC analysis in clinic routine. Therefore, in the context of the CANCER-ID consortium, we established a platform to assess technical validity of CTC detection methods in a European multi-center setting using non-small cell lung cancer (NSCLC) as a model. METHODS: We characterized multiple NSCLC cell lines to define cellular models distinct in their phenotype and molecular characteristics. Standardized tumor-cell-bearing blood samples were prepared at a central laboratory and sent to multiple European laboratories for processing according to standard operating procedures. The data were submitted via an online tool and centrally evaluated. Five CTC-enrichment technologies were tested. RESULTS: We could identify 2 cytokeratin expressing cell lines with distinct levels of EpCAM expression: NCI-H441 (EpCAMhigh, CKpos) and NCI-H1563 (EpCAMlow, CKpos). Both spiked tumor cell lines were detected by all technologies except for the CellSearch system that failed to enrich EpCAMlow NCI-H1563 cells. Mean recovery rates ranged between 49% and 75% for NCI-H411 and 32% and 76% for NCI-H1563 and significant differences were observed between the tested methods. CONCLUSIONS: This multi-national proficiency testing of CTC-enrichment technologies has importance in the establishment of guidelines for clinically applicable (pre)analytical workflows and the definition of minimal performance qualification requirements prior to clinical validation of technologies. It will remain in operation beyond the funding period of CANCER-ID in the context of the European Liquid Biopsy Society (ELBS).

Parkinson's disease-associated alterations of the gut microbiome predict disease-relevant changes in metabolic functions.

BACKGROUND: Parkinson's disease (PD) is a systemic disease clinically defined by the degeneration of dopaminergic neurons in the brain. While alterations in the gut microbiome composition have been reported in PD, their functional consequences remain unclear. Herein, we addressed this question by an analysis of stool samples from the Luxembourg Parkinson's Study (n = 147 typical PD cases, n = 162 controls). RESULTS: All individuals underwent detailed clinical assessment, including neurological examinations and neuropsychological tests followed by self-reporting questionnaires. Stool samples from these individuals were first analysed by 16S rRNA gene sequencing. Second, we predicted the potential secretion for 129 microbial metabolites through personalised metabolic modelling using the microbiome data and genome-scale metabolic reconstructions of human gut microbes. Our key results include the following. Eight genera and seven species changed significantly in their relative abundances between PD patients and healthy controls. PD-associated microbial patterns statistically depended on sex, age, BMI, and constipation. Particularly, the relative abundances of Bilophila and Paraprevotella were significantly associated with the Hoehn and Yahr staging after controlling for the disease duration. Furthermore, personalised metabolic modelling of the gut microbiomes revealed PD-associated metabolic patterns in the predicted secretion potential of nine microbial metabolites in PD, including increased methionine and cysteinylglycine. The predicted microbial pantothenic acid production potential was linked to the presence of specific non-motor symptoms. CONCLUSION: Our results suggest that PD-associated alterations of the gut microbiome can translate into substantial functional differences affecting host metabolism and disease phenotype.

Multicenter Evaluation of Circulating Cell-Free DNA Extraction and Downstream Analyses for the Development of Standardized (Pre)analytical Work Flows.

BACKGROUND: In cancer patients, circulating cell-free DNA (ccfDNA) can contain tumor-derived DNA (ctDNA), which enables noninvasive diagnosis, real-time monitoring, and treatment susceptibility testing. However, ctDNA fractions are highly variable, which challenges downstream applications. Therefore, established preanalytical work flows in combination with cost-efficient and reproducible reference materials for ccfDNA analyses are crucial for analytical validity and subsequently for clinical decision-making. METHODS: We describe the efforts of the Innovative Medicines Initiative consortium CANCER-ID (http://www.cancer-id.eu) for comparing different technologies for ccfDNA purification, quantification, and characterization in a multicenter setting. To this end, in-house generated mononucleosomal DNA (mnDNA) from lung cancer cell lines carrying known TP53 mutations was spiked in pools of plasma from healthy donors generated from 2 different blood collection tubes (BCTs). ccfDNA extraction was performed at 15 partner sites according to their respective routine practice. Downstream analysis of ccfDNA with respect to recovery, integrity, and mutation analysis was performed centralized at 4 different sites. RESULTS: We demonstrate suitability of mnDNA as a surrogate for ccfDNA as a process quality control from nucleic acid extraction to mutation detection. Although automated extraction protocols and quantitative PCR-based quantification methods yielded the most consistent and precise results, some kits preferentially recovered spiked mnDNA over endogenous ccfDNA. Mutated TP53 fragments derived from mnDNA were consistently detected using both next-generation sequencing-based deep sequencing and droplet digital PCR independently of BCT. CONCLUSIONS: This comprehensive multicenter comparison of ccfDNA preanalytical and analytical work flows is an important contribution to establishing evidence-based guidelines for clinically feasible (pre)analytical work flows.

Multicenter Evaluation of Circulating Plasma MicroRNA Extraction Technologies for the Development of Clinically Feasible Reverse Transcription Quantitative PCR and Next-Generation Sequencing Analytical Work Flows.

BACKGROUND: In human body fluids, microRNA (miRNA) can be found as circulating cell-free miRNA (cfmiRNA), as well as secreted into extracellular vesicles (EVmiRNA). miRNAs are being intensively evaluated as minimally invasive liquid biopsy biomarkers in patients with cancer. The growing interest in developing clinical assays for circulating miRNA necessitates careful consideration of confounding effects of preanalytical and analytical parameters. METHODS: By using reverse transcription quantitative real-time PCR and next-generation sequencing (NGS), we compared extraction efficiencies of 5 different protocols for cfmiRNA and 2 protocols for EVmiRNA isolation in a multicentric manner. The efficiency of the different extraction methods was evaluated by measuring exogenously spiked cel-miR-39 and 6 targeted miRNAs in plasma from 20 healthy individuals. RESULTS: There were significant differences between the tested methods. Although column-based extraction methods were highly effective for the isolation of endogenous miRNA, phenol extraction combined with column-based miRNA purification and ultracentrifugation resulted in lower quality and quantity of isolated miRNA. Among all extraction methods, the ubiquitously expressed miR-16 was represented with high abundance when compared with other targeted miRNAs. In addition, the use of miR-16 as an endogenous control for normalization of quantification cycle values resulted in a decreased variability of column-based cfmiRNA extraction methods. Cluster analysis of normalized NGS counts clearly indicated a method-dependent bias. CONCLUSIONS: The choice of plasma miRNA extraction methods affects the selection of potential miRNA marker candidates and mechanistic interpretation of results, which should be done with caution, particularly across studies using different protocols.

Does vacuum centrifugal concentration reduce yield or quality of nucleic acids extracted from FFPE biospecimens?

Vacuum centrifugal (SpeedVac) concentration is commonly applied to nucleic acids extracted from formalin-fixed paraffin-embedded (FFPE) sections, but with an unknown impact. We concentrated DNA and RNA from FFPE biospecimens using different time/temperature SpeedVac combinations of up to 30 min concentration at 45 °C, then used spectrophotometry, spectrofluorometry, RIN, PERM, DV200, qRT-PCR, DIN and the Illumina FFPE QC Assay to assess the changes in quantity, purity and integrity induced by the concentration process. We found the effects of SpeedVac concentration to be inconsequential, but an aliquot of elution buffer should be concentrated for use as the blank in spectrophotometry assays.

Preanalytical robustness of blood collection tubes with RNA stabilizers.

Background Efficient blood stabilization is essential to obtaining reliable and comparable RNA analysis data in preclinical operations. PAXgene (Qiagen, Becton Dickinson) and Tempus (Applied Biosystems, Life Technologies) blood collection tubes with RNA stabilizers both avoid preanalytical degradation of mRNA by endogenous nucleases and modifications in specific mRNA concentrations by unintentional up- or down-regulation of gene expression. Methods Sixteen different preanalytical conditions were tested in PAXgene and Tempus blood samples from seven donors: different mixing after collection, different fill volumes and different 24-h transport temperature conditions after collection. RNA was extracted by column-based methods. The quality of the extracted RNA was assessed by spectrophotometric quantification, A260/A280 purity ratio, RNA Integrity Number (Agilent Bioanalyzer), miRNA quantative real time polymerase chain reaction (qRT-PCR) on two target miRNAs (RNU-24 and miR-16), mRNA quality index by qRT-PCR on the 3' and 5' region of the GAPDH gene, and the PBMC preanalytical score, based on the relative expression levels of the IL8 and EDEM3 coding genes. Results When PAXgene RNA and Tempus blood collection tubes were used following the manufacturers' instructions, there was no statistically or technically significant difference in the output RNA quality attributes. However, the integrity of the RNA extracted from Tempus collection tubes was more sensitive to fill volumes and effective inversion, than to storage temperature, while the integrity of RNA extracted from PAXgene collection tubes was more sensitive to effective inversion and storage temperature than to fill volumes. Conclusions Blood collection tubes with different RNA stabilizers present different robustness to common preanalytical variations.

Preanalytical challenges - time for solutions.

The European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) Working Group for the Preanalytical Phase (WG-PRE) was originally established in 2013, with the main aims of (i) promoting the importance of quality in the preanalytical phase of the testing process, (ii) establishing best practices and providing guidance for critical activities in the preanalytical phase, (iii) developing and disseminating European surveys for exploring practices concerning preanalytical issues, (iv) organizing meetings, workshops, webinars or specific training courses on preanalytical issues. As education is a core activity of the WG-PRE, a series of European conferences have been organized every second year across Europe. This collective article summarizes the leading concepts expressed during the lectures of the fifth EFLM Preanalytical Conference "Preanalytical Challenges - Time for solutions", held in Zagreb, 22-23 March, 2019. The topics covered include sample stability, preanalytical challenges in hematology testing, feces analysis, bio-banking, liquid profiling, mass spectrometry, next generation sequencing, laboratory automation, the importance of knowing and measuring the exact sampling time, technology aids in managing inappropriate utilization of laboratory resources, management of hemolyzed samples and preanalytical quality indicators.

IL8 and IL16 levels indicate serum and plasma quality.

BACKGROUND: Longer pre-centrifugation times alter the quality of serum and plasma samples. Markers for such delays in sample processing and hence for the sample quality, have been identified. METHODS: Twenty cytokines in serum, EDTA plasma and citrate plasma samples were screened for changes in concentration induced by extended blood pre-centrifugation delays at room temperature. The two cytokines that showed the largest changes were further validated for their "diagnostic performance" in identifying serum or plasma samples with extended pre-centrifugation times. RESULTS: In this study, using R&D Systems ELISA kits, EDTA plasma samples and serum samples with a pre-centrifugation delay longer than 24 h had an IL16 concentration higher than 313 pg/mL, and an IL8 concentration higher than 125 pg/mL, respectively. EDTA plasma samples with a pre-centrifugation delay longer than 48 h had an IL16 concentration higher than 897 pg/mL, citrate plasma samples had an IL8 concentration higher than 21.5 pg/mL and serum samples had an IL8 concentration higher than 528 pg/mL. CONCLUSIONS: These robust and accurate tools, based on simple and commercially available ELISA assays can greatly facilitate qualification of serum and plasma legacy collections with undocumented pre-analytics.

Distinct metabolomic signature in cerebrospinal fluid in early parkinson's disease.

OBJECTIVE: The purpose of this study was to profile cerebrospinal fluid (CSF) from early-stage PD patients for disease-related metabolic changes and to determine a robust biomarker signature for early-stage PD diagnosis. METHODS: By applying a non-targeted and mass spectrometry-driven approach, we investigated the CSF metabolome of 44 early-stage sporadic PD patients yet without treatment (DeNoPa cohort). We compared all detected metabolite levels with those measured in CSF of 43 age- and gender-matched healthy controls. After this analysis, we validated the results in an independent PD study cohort (Tübingen cohort). RESULTS: We identified that dehydroascorbic acid levels were significantly lower and fructose, mannose, and threonic acid levels were significantly higher (P < .05) in PD patients when compared with healthy controls. These changes reflect pathological oxidative stress responses, as well as protein glycation/glycosylation reactions in PD. Using a machine learning approach based on logistic regression, we successfully predicted the origin (PD patients vs healthy controls) in a second (n = 18) as well as in a third and completely independent validation set (n = 36). The biomarker signature is composed of the three markers-mannose, threonic acid, and fructose-and allows for sample classification with a sensitivity of 0.790 and a specificity of 0.800. CONCLUSION: We identified PD-specific metabolic changes in CSF that were associated with antioxidative stress response, glycation, and inflammation. Our results disentangle the complexity of the CSF metabolome to unravel metabolome changes related to early-stage PD. The detected biomarkers help understanding PD pathogenesis and can be applied as biomarkers to increase clinical diagnosis accuracy and patient care in early-stage PD. © 2017 International Parkinson and Movement Disorder Society.

Ultraviolet C radiation influences the robustness of RNA integrity measurement.

The analytical and clinical validity of analyses of RNA samples destined for clinical diagnosis and therapeutic management is directly impacted by RNA quality. RNA is affected by heat, enzymatic degradation, and Ultraviolet (UV) light. RNA from three eukaryotic cell lines was degraded by heat, RNase, or UV light. RNA integrity values obtained with the benchmark Agilent Bioanalyzer 2100 system were compared with those from the more recent QIAxcel Advanced system. The application of this novel method has allowed us to unravel differences between RNA biophysical and biochemical degradation modes. Agilent RNA integrity number (RIN) and QIAxcel RIS were comparable in heat-degraded and RNase III-degraded RNA. Agilent RIN and QIAxcel RIS were comparable at a RIN decision level of 7 in UV-degraded RNA but not overall. The QIAxcel RIS method was more precise than Agilent RIN for RIN<8, while the inverse was true for RIN≥8. Greater degradation of mRNA and rRNA in UV-damaged samples hampered the Agilent RIN calculation algorithm. Overall, RIS was more robust than RIN for assessing RNA integrity. The ΔΔCt-values for heat- and UV-degraded RNA samples showed slightly higher correlation with RIS than with RIN. RNA integrity can be used to categorize RNA samples for suitability for downstream gene expression analyses, independently of the RNA degradation mechanism. The new method QIAxcel is more robust and therefore allows more accurate categorization of compromised RNA samples.

Replacing ß-mercaptoethanol in RNA extractions.

RNA extractions are potentially compromised in terms of both yield and quality by ribonucleases (RNases). The pungent and toxic reducing agent ß-mercaptoethanol (ß-ME), therefore, is commonly added to the biospecimen's lysis buffer to aid in RNase deactivation. Using different tissue types (liver tissue, kidney tissue, and cell pellets), extraction kits (RNeasy Mini Kit, Illustra RNA Spin Mini Kit, and PureLink Mini Kit), RNA quality assays (RNA integrity numbers [RINs] and quantitative real-time polymerase chain reaction [qRT-PCR]), yield assessments, and in vitro functional RNase assays (RNaseAlert Kit), we demonstrate that ß-ME should be replaced by the less toxic dithiothreitol (DTT) alternative.

Creation of a federated database of blood proteins: a powerful new tool for finding and characterizing biomarkers in serum.

Protein biomarkers offer major benefits for diagnosis and monitoring of disease processes. Recent advances in protein mass spectrometry make it feasible to use this very sensitive technology to detect and quantify proteins in blood. To explore the potential of blood biomarkers, we conducted a thorough review to evaluate the reliability of data in the literature and to determine the spectrum of proteins reported to exist in blood with a goal of creating a Federated Database of Blood Proteins (FDBP). A unique feature of our approach is the use of a SQL database for all of the peptide data; the power of the SQL database combined with standard informatic algorithms such as BLAST and the statistical analysis system (SAS) allowed the rapid annotation and analysis of the database without the need to create special programs to manage the data. Our mathematical analysis and review shows that in addition to the usual secreted proteins found in blood, there are many reports of intracellular proteins and good agreement on transcription factors, DNA remodelling factors in addition to cellular receptors and their signal transduction enzymes. Overall, we have catalogued about 12,130 proteins identified by at least one unique peptide, and of these 3858 have 3 or more peptide correlations. The FDBP with annotations should facilitate testing blood for specific disease biomarkers.

Implementing routine monitoring for nuclease contamination of equipment and consumables into the quality Management system of a laboratory.

Nucleases are ubiquitous in the environment, present in biospecimens and widely used in many laboratory processes. However, in the wrong context, as contaminants, they have catastrophic potential because of their ability to rapidly degrade nucleic acids whilst retaining high resilience to inactivation. Although laboratories undertake rigorous precautions to prevent nuclease contamination, such measures are not infallible. In 2015, we devised and integrated a novel routine nuclease testing regimen into our Quality Management System that uses cleavable, fluorescent DNA and RNA substrates to detect, monitor and control for nuclease contamination in our laboratory processes, equipment and consumables. The testing regimen enables us to identify higher-risk activities, design our laboratory workflows such that risk is minimized and help fulfil our obligations in respect of ISO 20387:2018 General Requirements for Biobanking and ISO 17025 Testing and Calibrations Laboratory standards, both of which stipulate that environmental conditions in our laboratory must be monitored with defined quality control criteria. In seventeen rounds of testing (30 Test Items per round), 1.1 % of RNase tests and 0.2 % of DNase tests returned elevated nuclease levels (≥2.90 x 10-9 U RNase or 1.67 x 10-3 U DNase) and we were able to take remedial action. In no instance was an elevated nuclease level consequential in terms of an impact on sample quality. We present our protocols, results and observations.

Stability and Qualification of a Legacy Fungal Collection.

Background: Microbial culture collections are valuable repositories for qualified and diverse microorganisms, playing a pivotal role in research, education, innovation, as well as in our response to current and emerging public health and societal challenges. However, such precious holdings, when not integrated in professional biobank infrastructures, may be vulnerable to major risks such as staff retirement, changes in the institutional strategy, or natural disasters. The process of preserving and rescuing "historical" collections can be long and treacherous with a loss of a part of the collection. At the Biological Resource Center of Institut Pasteur, we undertook the challenge of rescuing the dormant legacy fungal collection. Materials and Methods: A total of 64 freeze-dried strains, including yeasts and filamentous fungi, were characterized by using a polyphasic approach combining morphological features and molecular data. We assessed the viability, purity, and authenticity of selected strains isolated from multiple sources and stored for more than 20 years. Results: Our preliminary results show long-term stability of the selected strains and successful qualification in terms of purity and authentication. Moreover, based on the most recent taxonomic revisions, we updated and revised the nomenclature, where applicable. Conclusion: Our findings demonstrated the potential value of reviving historical microbial collections for biobanking and research activities and reassure us about the collection's future reopening.

A Ranking Tool for "Category Killer" Microbial Biobanks.

Microbial biobanks preserve and provide microbial bioresources for research, training, and quality control purposes. They ensure the conservation of biodiversity, contribute to taxonomical research, and support scientific advancements. Microbial biobanks can cover a wide range of phylogenetic and metabolic diversity ("category killers") or focus on specific taxonomic, thematic, or disease areas. The strategic decisions about strain selection for certain applications or for the biobank culling necessitate a method to support prioritization and selection. Here, we propose an unbiased scoring approach based on objective parameters to assess, categorize, and assign priorities among samples in stock in a microbial biobank. We describe the concept of this ranking tool and its application to identify high-priority strains for whole genome sequencing with two main goals: (i) genomic characterization of quality control, reference, and type strains; (ii) genome mining for the discovery of natural products, bioactive and antimicrobial molecules, with focus on human diseases. The general concept of the tool can be useful to any biobank and for any ranking or culling needs.

Toward a common standard for data and specimen provenance in life sciences.

Open and practical exchange, dissemination, and reuse of specimens and data have become a fundamental requirement for life sciences research. The quality of the data obtained and thus the findings and knowledge derived is thus significantly influenced by the quality of the samples, the experimental methods, and the data analysis. Therefore, a comprehensive and precise documentation of the pre-analytical conditions, the analytical procedures, and the data processing are essential to be able to assess the validity of the research results. With the increasing importance of the exchange, reuse, and sharing of data and samples, procedures are required that enable cross-organizational documentation, traceability, and non-repudiation. At present, this information on the provenance of samples and data is mostly either sparse, incomplete, or incoherent. Since there is no uniform framework, this information is usually only provided within the organization and not interoperably. At the same time, the collection and sharing of biological and environmental specimens increasingly require definition and documentation of benefit sharing and compliance to regulatory requirements rather than consideration of pure scientific needs. In this publication, we present an ongoing standardization effort to provide trustworthy machine-actionable documentation of the data lineage and specimens. We would like to invite experts from the biotechnology and biomedical fields to further contribute to the standard.

New RT-PCR Assay for the Detection of Current and Future SARS-CoV-2 Variants.

Multiple lineages of SARS-CoV-2 have been identified featuring distinct sets of genetic changes that confer to the virus higher transmissibility and ability to evade existing immunity. The continuous evolution of SARS-CoV-2 may pose challenges for current treatment options and diagnostic tools. In this study, we have first evaluated the performance of the 14 WHO-recommended real-time reverse transcription (RT)-PCR assays currently in use for the detection of SARS-CoV-2 and found that only one assay has reduced performance against Omicron. We then developed a new duplex real-time RT-PCR assay based on the amplification of two ultra-conserved elements present within the SARS-CoV-2 genome. The new duplex assay successfully detects all of the tested SARS-CoV-2 variants of concern (including Omicron sub-lineages BA.4 and BA.5) from both clinical and wastewater samples with high sensitivity and specificity. The assay also functions as a one-step droplet digital RT-PCR assay. This new assay, in addition to clinical testing, could be adopted in surveillance programs for the routine monitoring of SARS-CoV-2's presence in a population in wastewater samples. Positive results with our assay in conjunction with negative results from an Omicron-specific assay may provide timely indication of the emergence of a novel SARS-CoV-2 variant in a certain community and thereby aid public health interventions.

Biospecimen Qualification in a Clinical Biobank of Urological Diseases.

Development of novel biomarkers for diagnosis of disease and assessment of treatment efficacy utilizes a wide range of biospecimens for discovery research. The fitness of biospecimens for the purpose of biomarker development depends on the clinical characteristics of the donor and on a number of critical and potentially uncontrolled pre-analytical variables. Pre-analytical factors influence the reliability of the biomarkers to be analyzed and can seriously impact analytic outcomes. Sample quality stratification assays and tools can be utilized by biorepositories to minimize bias resulting from samples' inconsistent quality. In this study, we evaluated the quality of biobanked specimens by comparing analytical outcomes at 1, 5, and 10 years after collection. Our results demonstrate that currently available assays and tools can be used by biobank laboratories to support objective biospecimen qualification. We have established a workflow to monitor the quality of different types of biospecimens and, in this study, present the results of a qualification exercise applied to fluid samples and their derivatives in the context of urological diseases.