Dataset Information

SPIDIA-RNA: second external quality assessment for the pre-analytical phase of blood samples used for RNA based analyses.

ABSTRACT: One purpose of the EC funded project, SPIDIA, is to develop evidence-based quality guidelines for the pre-analytical handling of blood samples for RNA molecular testing. To this end, two pan-European External Quality Assessments (EQAs) were implemented. Here we report the results of the second SPIDIA-RNA EQA. This second study included modifications in the protocol related to the blood collection process, the shipping conditions and pre-analytical specimen handling for participants. Participating laboratories received two identical proficiency blood specimens collected in tubes with or without an RNA stabilizer. For pre-defined specimen storage times and temperatures, laboratories were asked to perform RNA extraction from whole blood according to their usual procedure and to return extracted RNA to the SPIDIA facility for further analysis. These RNA samples were evaluated for purity, yield, integrity, stability, presence of interfering substances, and gene expression levels for the validated markers of RNA stability: FOS, IL1B, IL8, GAPDH, FOSB and TNFRSF10c. Analysis of the gene expression results of FOS, IL8, FOSB, and TNFRSF10c, however, indicated that the levels of these transcripts were significantly affected by blood collection tube type and storage temperature. These results demonstrated that only blood collection tubes containing a cellular RNA stabilizer allowed reliable gene expression analysis within 48 h from blood collection for all the genes investigated. The results of these two EQAs have been proposed for use in the development of a Technical Specification by the European Committee for Standardization.

SUBMITTER: Malentacchi F

PROVIDER: S-EPMC4226503 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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SPIDIA-RNA: second external quality assessment for the pre-analytical phase of blood samples used for RNA based analyses.

Malentacchi Francesca F Pazzagli Mario M Simi Lisa L Orlando Claudio C Wyrich Ralf R Günther Kalle K Verderio Paolo P Pizzamiglio Sara S Ciniselli Chiara Maura CM Zhang Hui H Korenková Vlasta V Rainen Lynne L Bar Tzachi T Kubista Mikael M Gelmini Stefania S

PloS one 20141110 11

One purpose of the EC funded project, SPIDIA, is to develop evidence-based quality guidelines for the pre-analytical handling of blood samples for RNA molecular testing. To this end, two pan-European External Quality Assessments (EQAs) were implemented. Here we report the results of the second SPIDIA-RNA EQA. This second study included modifications in the protocol related to the blood collection process, the shipping conditions and pre-analytical specimen handling for participants. Participatin ...[more]

PMID: 25384019

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Project description:BackgroundPre-analytical conditions are key factors in maintaining the high quality of biospecimens. They are necessary for accurate reproducibility of experiments in the field of biomarker discovery as well as achieving optimal specificity of laboratory tests for clinical diagnosis. In research at the National Biobank of Korea, we evaluated the impact of pre-analytical conditions on the stability of biobanked blood samples by measuring biochemical analytes commonly used in clinical laboratory tests.MethodsWe measured 10 routine laboratory analytes in serum and plasma samples from healthy donors (n = 50) with a chemistry autoanalyzer (Hitachi 7600-110). The analyte measurements were made at different time courses based on delay of blood fractionation, freezing delay of fractionated serum and plasma samples, and at different cycles (0, 1, 3, 6, 9) of freeze-thawing. Statistically significant changes from the reference sample mean were determined using the repeated-measures ANOVA and the significant change limit (SCL).ResultsThe serum levels of GGT and LDH were changed significantly depending on both the time interval between blood collection and fractionation and the time interval between fractionation and freezing of serum and plasma samples. The glucose level was most sensitive only to the elapsed time between blood collection and centrifugation for blood fractionation. Based on these findings, a simple formula (glucose decrease by 1.387 mg/dL per hour) was derived to estimate the length of time delay after blood collection. In addition, AST, BUN, GGT, and LDH showed sensitive responses to repeated freeze-thaw cycles of serum and plasma samples.ConclusionThese results suggest that GGT and LDH measurements can be used as quality control markers for certain pre-analytical conditions (eg, delayed processing or repeated freeze-thawing) of blood samples which are either directly used in the laboratory tests or stored for future research in the biobank.

Project description:Zinc deficiency continues to be a major concern for global public health. The zinc status of a target population is typically estimated by measuring circulating zinc levels, but the sampling procedures are not standardized and thus may result in analytical discrepancies. To examine this, we designed a study that controlled most of the technical parameters in order to focus on five pre-analytical variables reported to influence the measurement of zinc in blood samples, including (1) blood draw site (capillary or venous), (2) blood sample matrix (plasma or serum), (3) blood collection tube manufacturer (Becton, Dickinson and Company or Sarstedt AG & Co), (4) blood processing time (0, 4, or 24 hours), and (5) blood holding temperatures (4°C, 20°C, or 37°C). A diverse cohort of 60 healthy adults were recruited to provide sequential capillary and venous blood samples, which were carefully processed under a single chain of custody and measured for zinc content using inductively coupled plasma optical emission spectrometry. When comparing blood draw sites, the mean zinc content of capillary samples was 0.054 mg/L (8%; p<0.0001) higher than venous blood from the same donors. When comparing blood sample matrices, the mean zinc content of serum samples was 0.029 mg/L (5%; p<0.0001) higher than plasma samples from the same donors. When comparing blood collection tube manufacturer, the mean zinc content from venous blood samples did not differ between venders, but the mean zinc content from BD capillary plasma was 0.036 mg/L (6%; p<0.0001) higher than Sarstedt capillary plasma from the same donors. When comparing processing times, the mean zinc content of plasma and serum samples was 5-12% higher (p<0.0001) in samples processed 4-24 hour after collection. When comparing holding temperatures, the mean zinc content of plasma and serum samples was 0.5-7% higher (p = 0.0007 or p = 0.0061, respectively) in samples temporarily held at 20°C or 37°C after collection. Thus even with the same donors and blood draws, significant differences in zinc content were observed with different draw sites, tube types, and processing procedures, demonstrating that key pre-analytic variables can have an impact on zinc measurement, and subsequent classification of zinc status. Minimizing these pre-analytical variables is important for generating best practice guidelines for assessment of zinc status.

Project description:Metabolomic and proteomic analyses of human plasma and serum samples harbor the power to advance our understanding of disease biology. Pre-analytical factors may contribute to variability and bias in the detection of analytes, especially when multiple labs are involved, caused by sample handling, processing time, and differing operating procedures. To better understand the impact of pre-analytical factors that are relevant to implementing a unified proteomic and metabolomic approach in a clinical setting, we assessed the influence of temperature, sitting times, and centrifugation speed on the plasma and serum metabolomes and proteomes from six healthy volunteers. We used targeted metabolic profiling (497 metabolites) and data-independent acquisition (DIA) proteomics (572 proteins) on the same samples generated with well-defined pre-analytical conditions to evaluate criteria for pre-analytical SOPs for plasma and serum samples. Time and temperature showed the strongest influence on the integrity of plasma and serum proteome and metabolome. While rapid handling and low temperatures (4°C) are imperative for metabolic profiling, the analyzed proteomics data set showed variability when exposed to temperatures of 4°C for more than 2 h, highlighting the need for compromises in a combined analysis. We formalized a quality control scoring system to objectively rate sample stability and tested this score using external data sets from other pre-analytical studies. Stringent and harmonized standard operating procedures (SOPs) are required for pre-analytical sample handling when combining proteomics and metabolomics of clinical samples to yield robust and interpretable data on a longitudinal scale and across different clinics. To ensure an adequate level of practicability in a clinical routine for metabolomics and proteomics studies, we suggest keeping blood samples up to 2 h on ice (4°C) prior to snap-freezing as a compromise between stability and operability. Finally, we provide the methodology as an open-source R package allowing the systematic scoring of proteomics and metabolomics data sets to assess the stability of plasma and serum samples.

Dataset Information

SPIDIA-RNA: second external quality assessment for the pre-analytical phase of blood samples used for RNA based analyses.

Publications

SPIDIA-RNA: second external quality assessment for the pre-analytical phase of blood samples used for RNA based analyses.

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