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.

Method Optimization of Skin Biopsy-Derived Fibroblast Culture for Reprogramming Into Induced Pluripotent Stem Cells.

Background: Fibroblasts can be isolated from skin biopsies using a chemical dissociation, a physical dissociation, or a combination of both techniques. They can be reprogrammed into induced pluripotent stem cells (iPSCs) through the introduction of defined sets of key transcription factors. This study aimed to identify the optimal protocol for skin biopsy dissociation, fibroblast culture, and fibroblast cryopreservation in the scope of reprogramming into iPSCs and in the context of biobank accreditation. Methods: First, four dissociation techniques typically used in the laboratory (explant based, enzymatic, and/or mechanical) and two cryopreservation media containing 10% dimethyl sulfoxide, either commercial or homemade, were evaluated in terms of post-thaw recovery, viability, growth curves, and karyotyping analyses of the fibroblasts. Next, the clones reprogrammed from the fibroblasts isolated with the two optimal dissociation methods and cryopreservation media were further assessed by reprogramming quality before cryopreservation and post-thaw pluripotency comparison. Results: Fibroblasts isolated from skin biopsies using an explant-based or enzymatic dissociation method showed higher viability, higher proliferative potential, and higher genome stability post-thaw compared to the other dissociation techniques. Fibroblasts obtained by the explant-based dissociation technique showed a slightly higher reprogramming quality. The iPSC reprogrammed from explant-based dissociated fibroblasts showed successful recovery of iPSC clones. No difference between the two cryopreservation media was detected for the tested endpoints, with the exception of a higher visual count of colonies at the end of the reprogramming for the explant-based dissociation method. Conclusions: This article presents a formal method optimization for biospecimen processing in the context of accreditation in laboratories and biobanks. We validated skin biopsy-derived fibroblast isolation, culture, and cryopreservation for downstream mRNA reprogramming into iPSCs. The explant-based dissociation technique and homemade medium are selected as optimal to isolate and cryopreserve fibroblasts from skin biopsies in the scope of reprogramming into iPSCs.

A Cystatin C Cleavage ELISA Assay as a Quality Control Tool for Determining Sub-Optimal Storage Conditions of Cerebrospinal Fluid Samples in Alzheimer's Disease Research.

BACKGROUND: An N-terminal octapeptide cleavage of the cystatin C protein was discovered by mass spectrometry when cerebrospinal fluid (CSF) was stored at -20°C for 3 months, which did not occur when CSF was stored at -80°C. OBJECTIVE: The aim was to develop an immunoassay as quality assessment tool to detect this -20°C cleavage of cystatin C in CSF and support Alzheimer's disease research. METHODS: A specific monoclonal antibody and a double indirect sandwich ELISA were developed: one assay quantifies the octapeptide uncleaved protein specifically and the other quantifies the total cystatin C present in the biological fluid (both cleaved and uncleaved forms). The ratio of these concentrations was calculated to assess the extent of cleavage of cystatin C. The novel ELISA was validated and applied in a short-term (up to 4 weeks) and mid-term (up to one year) stability study of CSF stored at 4°C, -20°C, -80°C, and liquid nitrogen. Impact of freeze-thaw cycles, adsorption, and protease inhibitors were tested. RESULTS: The ratio of truncated protein was modified following -20°C storage and seemed to reach a plateau after 6 months. The ratio was impacted neither by freeze-thaw cycles nor adsorption. The -20°C specific cleavage was found to be protease related. CONCLUSION: Using this novel double indirect sandwich ELISA, absolute levels of the total and uncleaved cystatin C and the ratio of truncated cystatin C can be measured. This assay is an easily applicable tool which can be used to confirm that CSF biospecimen are fit-for-purpose for Alzheimer's disease research.

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).

Optimization of Viable Glioblastoma Cryopreservation for Establishment of Primary Tumor Cell Cultures.

Background: Technologies related to the establishment of primary tumor cell cultures from solid tumors, including glioblastoma, are increasingly important to oncology research and practice. However, processing of fresh tumor specimens for establishment of primary cultures on the day of surgical collection is logistically difficult. The feasibility of viable cryopreservation of glioblastoma specimens, allowing for primary culture establishment weeks to months after surgical tumor collection and freezing, was demonstrated by Mullins et al. in 2013, with a success rate of 59% that was not significantly lower than that achieved with fresh tumor tissue. However, research targeting optimization of viable glioblastoma cryopreservation protocols for establishment of primary tumor cultures has been limited. Objectives: The objective of this study was to optimize glioblastoma cryopreservation methods for viable cryobanking and to determine if two-dimensional (2D) or three-dimensional (3D) culture conditions were more supportive of glioblastoma growth after thawing of frozen tumor specimens. Methods: Portions of eight human glioblastoma specimens were cryopreserved by four different protocols differing in the time of enzymatic digestion (before or after cryopreservation), and in the type of cryopreservation media (CryoStor CS10 or 10% dimethyl sulfoxide and 90% fetal calf serum). After 1 month, frozen tissues were thawed, enzymatically digested, if not digested before, and used for initiation of 2D or 3D primary tumor cultures to determine viability. Results: Among the tested cryopreservation and culturing protocols, the most efficient combinations of cryopreservation and culture were those associated with the use of CryoStor CS10 cryopreservation medium, enzymatic digestion before freezing, and 2D culturing after thawing with a successful culture rate of 8 out of 8 cases (100%). Two-dimensional cultures were in general more efficient for the support of tumor cell growth after thawing than 3D cultures. Conclusions: This study supports development of evidence-based viable glioblastoma cryopreservation methods for use in glioblastoma biobanking and research.

The European NAFLD Registry: A real-world longitudinal cohort study of nonalcoholic fatty liver disease.

Non-Alcoholic Fatty Liver Disease (NAFLD), a progressive liver disease that is closely associated with obesity, type 2 diabetes, hypertension and dyslipidaemia, represents an increasing global public health challenge. There is significant variability in the disease course: the majority exhibit only fat accumulation in the liver but a significant minority develop a necroinflammatory form of the disease (non-alcoholic steatohepatitis, NASH) that may progress to cirrhosis and hepatocellular carcinoma. At present our understanding of pathogenesis, disease natural history and long-term outcomes remain incomplete. There is a need for large, well characterised patient cohorts that may be used to address these knowledge gaps and to support the development of better biomarkers and novel therapies. The European NAFLD Registry is an international, prospectively recruited observational cohort study that aims to establish a large, highly-phenotyped patient cohort and linked bioresource. Here we describe the infrastructure, data management and monitoring plans, and the standard operating procedures implemented to ensure the timely and systematic collection of high-quality data and samples. Already recruiting subjects at secondary/tertiary care centres across Europe, the Registry is supporting the European Union IMI2-funded LITMUS 'Liver Investigation: Testing Marker Utility in Steatohepatitis' consortium, which is a major international effort to robustly validate biomarkers that diagnose, risk stratify and/or monitor NAFLD progression and liver fibrosis stage. The European NAFLD Registry has the demonstrable capacity to support research and biomarker development at scale and pace.

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.

Standardization of the preanalytical phase of DNA extraction from fixed tissue for next-generation sequencing analyses.

Next-generation sequencing (NGS) analyses on DNA derived from archived Formalin-Fixed Paraffin-Embedded (FFPE) clinical material can provide a powerful tool in oncology research and clinical diagnostics. Although several studies have established that NGS can be performed using DNA from FFPE tissue, the accuracy and reproducibility of such analyses, as well as their robustness to the biomolecular quality of the samples used, remains a matter of debate. Excellent reviews have recently been published, providing evidence-based best practices for FFPE DNA extraction. Alternative fixatives exist, although their implementation in clinical practice is difficult. In this article, we present (i) a review of fixed tissue DNA preanalytics with a special focus on DNA extraction and fixed tissue sample qualification and (ii) results from comparisons between different methods of DNA extraction from tissue samples that have been fixed or stabilized by different methods, in terms of NGS metrics and different DNA quality metrics.

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.

Cold Ischemia Score: An mRNA Assay for the Detection of Extended Cold Ischemia in Formalin-Fixed, Paraffin-Embedded Tissue.

Although there are thousands of formalin-fixed paraffin-embedded (FFPE) tissue blocks potentially available for scientific research, many are of questionable quality, partly due to unknown preanalytical variables. We analyzed FFPE tissue biospecimens as part of the National Cancer Institute (NCI) Biospecimen Preanalytical Variables program to identify mRNA markers denoting cold ischemic time. The mRNA was extracted from colon, kidney, and ovary cancer FFPE blocks (40 patients, 10-12 hr fixation time) with 1, 2, 3, and 12 hr cold ischemic times, then analyzed using qRT-PCR for 23 genes selected following a literature search. No genes tested could determine short ischemic times (1-3 hr). However, a combination of three unstable genes normalized to a more stable gene could generate a "Cold Ischemia Score" that could distinguish 1 to 3 hr cold ischemia from 12 hr cold ischemia with 62% sensitivity and 84% specificity.

Small Nucleolar RNA Score: An Assay to Detect Formalin-Overfixed Tissue.

Although there are millions of formalin-fixed paraffin-embedded (FFPE) tissue blocks potentially available for scientific research, many are of questionable quality, partly due to unknown fixation conditions. We analyzed FFPE tissue biospecimens as part of the NCI Biospecimen Preanalytical Variables (BPV) program to identify microRNA (miRNA) markers for fixation time. miRNA was extracted from kidney and ovary tumor FFPE blocks (19 patients, cold ischemia ≤2 hours) with 6, 12, 24, and 72 hours fixation times, then analyzed using the WaferGen SmartChip platform (miRNA chip with 1036 miRNA targets). For fixation time, principal component analysis of miRNA chip expression data separated 72 hours fixed samples from 6 to 24 hours fixed samples. A set of small nuclear RNA (snRNA) targets was identified that best determines fixation time and was validated using a second independent cohort of seven different tissue types. A customized assay was then developed, based on a set of 24 miRNA and snRNA targets, and a simple "snoRNA score" defined. This score detects FFPE tissue samples with fixation for 72 hours or more, with 79% sensitivity and 80% specificity. It can therefore be used to assess the fitness-for-purpose of FFPE samples for DNA or RNA-based research or clinical assays, which are known to be of limited robustness to formalin overfixation.

Biospecimens and Biobanking in Global Health.

Biobanks provide a critical infrastructure to support research in human health. Biospecimens and their accompanying data are increasingly needed to support biomedical research and clinical care. The original text was initially published in the Handbook for Cancer Research in Africa. The value of this publication is great as it underlines the importance of biobanks in Africa as a key resource to increase quality scientific research and participate in global health research. Therefore, a revision to extend these principles to other low resource contexts, to include updated material and references and add the topic of biobank sustainability were relevant.

Preanalytical Stability of Antibodies to Pathogenic Antigens.

Background: Serologic testing for antibodies against epitopes from pathogens is a valuable tool for investigating the relationship between infection and disease. This study comprehensively evaluates the impact of preanalytic variation on antibody seropositivities to a selected set of antigens arising from delays in processing of blood samples, preprocessing storage temperature, and vacutainer type.Methods: We assessed peripheral blood collected from 29 volunteers in four different Vacutainer types [ethylenediaminoetetraacetic acid (EDTA), acid-citrate-dextrose (ACD), lithium heparin (LH), serum separator tubes (SST)], and stored at 4°C or room temperature for 0, 1, 2, 3, 4, 5, and 6 days before processing. Multiplex serology was used to determine antibody reactivity against 35 antigens derived from human papillomaviruses, human polyomaviruses, Epstein-Barr virus, and Helicobacter pylori Cohen's κ statistic was used to measure agreement on seropositivity status between samples exposed to standard and nonstandard clinical practice conditions.Results: For samples processed without delay, κ was not associated with storage-temperature (P value range 0.23 to 0.95) or vacutainer type (P value range, 0.35-0.89). Kappa did not significantly decline with increasing delays in processing for any vacutainer-type storage temperature combination (P slope range, 0.06-1.00).Conclusions: Antibodies to epitopes from various pathogenic infectious agents can be measured reliably from samples stored in SST, EDTA, ACD, or LH vacutainers at either room temperature or 4°C for up to 6 days before processing.Impact: Serologic testing is robust to several preanalytic options. These findings are particularly important for epidemiologic studies recruiting participants from remote settings where sample exposure to preanalytic conditions can vary considerably. Cancer Epidemiol Biomarkers Prev; 26(8); 1337-44. ©2017 AACR.

A Critical Evaluation of the PAXgene Tissue Fixation System: Morphology, Immunohistochemistry, Molecular Biology, and Proteomics.

OBJECTIVES: To evaluate the PAXgene tissue fixation system. METHODS: Clinical biospecimens (n = 46) were divided into PAXgene-fixed paraffin-embedded (PFPE), formalin-fixed paraffin-embedded (FFPE), and fresh-frozen (FF) blocks. PFPE and FFPE sections were compared for histology (H&E staining) and immunohistochemistry (14 antibodies) using tissue microarrays. PFPE, FFPE, and FF samples were compared in terms of RNA quality (RNA integrity number, polymerase chain reaction [PCR] amplicon length, and quantitative reverse transcription PCR), DNA quality (gel electrophoresis and methylation profiling) and protein quality (liquid chromatography-mass spectrometry [LC-MS/MS]). RESULTS: PFPE protocol optimization was required in most cases and is described. RNA extracted from PFPE sections was considerably less degraded than that from FFPE sections but more degraded than that from FF blocks. Genomic-length DNA was extracted from PFPE and FF biospecimens, and methylation profiling showed PFPE and FF biospecimens to be almost indistinguishable. Only degraded DNA was extracted from FFPE biospecimens. PFPE sections yielded peptides that were slightly less amenable to LC-MS/MS analysis than FFPE sections, but FF gave slightly better results. CONCLUSIONS: While it cannot be envisaged that PAXgene will replace formalin in a routine clinical setting, for specific projects or immunodiagnostics involving biospecimens destined for immunohistochemical or histologic staining and DNA or RNA analyses, PAXgene is a viable option.

The effect of long-term -80°C storage of thyroid biospecimens on RNA quality and ensuring fitness for purpose.

AIMS: To establish whether RNA degrades in long-term storage at -80°C and whether RNA integrity numbers (RINs) determine 'fitness for purpose' in severely degraded RNA. METHODS: RNA was extracted from 549 thyroid biospecimens stored at -80°C for 0.1-10.9 years then their RINs correlated with storage time. RT-PCR for 65, 265, 534 and 942 base pair amplicons of hydroxymethylbilane synthase was used to measure amplicon length in RNA from cryopreserved and FFPE biospecimens that were equally degraded according to RIN. RESULTS: Storage time did not correlate with RIN. Longer amplicons were obtained from cryopreserved samples than FFPE samples with equal RINs. CONCLUSIONS: RNA does not degrade in thyroid biospecimens stored for long periods of time at -80°C. Although RINs are known to predict amenability to analytical platforms in good quality samples, this prediction is unreliable in severely degraded samples.

An Independent Evaluation of the CryoXtract Instruments' CXT350 Frozen Sample Aliquotter Using Tissue and Fecal Biospecimens.

The ability to take targeted multiple cores from a single frozen biospecimen would enable several research projects to be fueled from one biospecimen, a small piece of tissue to be quality-control tested, and for pathologically-discrete areas of a biospecimen (e.g., tumor, stromal, and normal tissue) to be selectively sampled for comparative analyses. CryoXtract Instruments' CXT350 Frozen Sample Aliquotter can potentially achieve this by producing multiple cores from one cryopreserved biospecimen without thawing either the parent biospecimen or its daughter cores. It therefore has the potential to add significant value to a tissue banking workflow. We have evaluated its performance while using 614 cores from fecal, liver, kidney, lung, heart, and colon biospecimens. Coring densities of up to five complete and four fragmentary cores per cm(3) are achievable using 3 mm coring probes. Median core weights for tissue were 14.1-17.2 mg (depending on tissue type) and cores ≤325 mg could be taken from fecal biospecimens (depending on the fill-depth of the tube). The coefficient of variation for multiple cores taken from a fecal biospecimen was 11.7%. Between-sample contamination did not occur. RNA Integrity numbers and qRT-PCR analysis demonstrated that coring induced a statistically significant impact on RNA quality that was inconsequential in magnitude and in our view does not represent a barrier for the effective utilization of the technology.

Tube Polypropylene: A Neglected Critical Parameter for Protein Adsorption During Biospecimen Storage.

This biospecimen research case study illustrates the importance of a neglected pre-analytical factor, the polypropylene type of storage tubes. We measured amyloid ß1-42 peptide and showed that a non-irradiated, homopolymer type of polypropylene has the lowest adsorption properties.

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.

Method validation for preparing urine samples for downstream proteomic and metabolomic applications.

BACKGROUND: Formal validation of methods for biospecimen processing in the context of accreditation in laboratories and biobanks is lacking. A protocol for processing of a biospecimen (urine) was validated for fitness-for-purpose in terms of key downstream endpoints. METHODS: Urine processing was optimized for centrifugation conditions on the basis of microparticle counts at room temperature (RT) and at 4°C. The optimal protocol was validated for performance (microparticle counts), and for reproducibility and robustness for centrifugation temperature (4°C vs. RT) and brake speed (soft, medium, hard). Acceptance criteria were based on microparticle counts, cystatin C and creatinine concentrations, and the metabolomic profile. RESULTS: The optimal protocol was a 20-min, 12,000 g centrifugation at 4°C, and was validated for urine collection in terms of microparticle counts. All reproducibility acceptance criteria were met. The protocol was robust for centrifugation at 4°C versus RT for all parameters. The protocol was considered robust overall in terms of brake speeds, although a hard brake gave significantly fewer microparticles than a soft brake. CONCLUSIONS: We validated a urine processing method suitable for downstream proteomic and metabolomic applications. Temperature and brake speed can influence analytic results, with 4°C and high brake speed considered optimal. Laboratories and biobanks should ensure these conditions are systematically recorded in the scope of accreditation.