Type Of Collection Tube Research Articles

A comparative evaluation of lithium estimation for samples collected in different tubes and its stability on storage.

PURPOSE:Lithium (Li) is a well-established drug for the treatment of bipolar affective disorders. Li as a drug is known to possess a narrow therapeutic index. Thus, regular monitoring of blood Li in patients receiving Li therapy is essential. Plain tubes with clot activator are known to interfere with Li estimation. The current study was planned to compare Li estimation in sera from vacutainers with clot activator, and plasma from sodium heparinized vacutainers with that of Li estimation in sera from glass vials. The time-dependant stability of Li estimation on storage at 2°C–8°C for 48 h in these three set of tubes was also evaluated.MATERIALS AND METHODS:Blood from the patients on Li therapy (n = 100) was collected in 3 different collection tubes: plain vacutainer with clot activator (S), Sodium heparinized vacutainer (P) and Glass vial (G) and was analyzed by ion selective electrode (ISE) analyzer for Li levels. Secondary aliquots were also taken from each type of collection tube and stored at 2°C–8°C. Time-dependant stability of Li estimation was checked at 12 h, 24 h, and 48 h. ANOVA followed by Tukey's posttest was performed to calculate statistical significance taking glass vial as reference collection tube. Bland–Altman plots were plotted to compare between three collection tubes at baseline. Stability on storage was defined when >95% of the samples were within allowable error limit for that time point taking baseline levels as reference.RESULTS:A mean bias of 0.18 mmol/L and mean percentage bias of 19.9% in Li levels was observed between serum from (S) than serum from (G). This difference was found to be statistically significant. However, statistically nonsignificant mean bias of 0.02 mmol/L and mean percentage bias of 3.34% in Li levels was observed between plasma from (P) and serum from (G). Time-dependant stability was observed more in glass vials as compared to vacutainers with clot activator or sodium heparin.CONCLUSION:Serum from glass vial should be the preferred method for blood collection to determine Li levels.

Effect of Blood Collection Tube Type and Time to Processing on the Enumeration and High-Content Characterization of Circulating Tumor Cells Using the High-Definition Single-Cell Assay.

- As circulating tumor cell (CTC) assays gain clinical relevance, it is essential to address preanalytic variability and to develop standard operating procedures for sample handling in order to successfully implement genomically informed, precision health care. - To evaluate the effects of blood collection tube (BCT) type and time-to-assay (TTA) on the enumeration and high-content characterization of CTCs by using the high-definition single-cell assay (HD-SCA). - Blood samples of patients with early- and advanced-stage breast cancer were collected into cell-free DNA (CfDNA), EDTA, acid-citrate-dextrose solution, and heparin BCTs. Time-to-assay was evaluated at 24 and 72 hours, representing the fastest possible and more routine domestic shipping intervals, respectively. - We detected the highest CTC levels and the lowest levels of negative events in CfDNA BCT at 24 hours. At 72 hours in this BCT, all CTC subpopulations were decreased with the larger effect observed in high-definition CTCs and cytokeratin-positive cells smaller than white blood cells. Overall cell retention was also optimal in CfDNA BCT at 24 hours. Whole-genome copy number variation profiles were generated from single cells isolated from all BCT types and TTAs. Cells from CfDNA BCT at 24-hour TTA exhibited the least noise. - Circulating tumor cells can be identified and characterized under a variety of collection, handling, and processing conditions, but the highest quality can be achieved with optimized conditions. We quantified performance differences of the HD-SCA for specific preanalytic variables that may be used as a guide to develop best practices for implementation into patient care and/or research biorepository processes.

Non-invasive fetal RHD genotyping for RhD negative women stratified into RHD gene deletion or variant groups: comparative accuracy using two blood collection tube types

Non-invasive fetal RHD genotyping in Australia to reduce anti-D usage will need to accommodate both prolonged sample transport times and a diverse population demographic harbouring a range of RHD blood group gene variants. We compared RHD genotyping accuracy using two blood sample collection tube types for RhD negative women stratified into deleted RHD gene haplotype and RHD gene variant cohorts. Maternal blood samples were collected into EDTA and cell-free (cf)DNA stabilising (BCT) tubes from two sites, one interstate. Automated DNA extraction and polymerase chain reaction (PCR) were used to amplify RHD exons 5 and 10 and CCR5. Automated analysis flagged maternal RHD variants, which were classified by genotyping. Time between sample collection and processing ranged from 2.9 to 187.5 hours. cfDNA levels increased with time for EDTA (range 0.03-138ng/μL) but not BCT samples (0.01-3.24ng/μL). For the 'deleted' cohort (n=647) all fetal RHD genotyping outcomes were concordant, excepting for one unexplained false negative EDTA sample. Matched against cord RhD serology, negative predictive values using BCT and EDTA tubes were 100% and 99.6%, respectively. Positive predictive values were 99.7% for both types. Overall 37.2% of subjects carried an RhD negative baby. The 'variant' cohort (n=15) included one novel RHD and eight hybrid or African pseudogene variants. Review for fetal RHD specific signals, based on one exon, showed three EDTA samples discordant to BCT, attributed to high maternal cfDNA levels arising from prolonged transport times. For the deleted haplotype cohort, fetal RHD genotyping accuracy was comparable for samples collected in EDTA and BCT tubes despite higher cfDNA levels in the EDTA tubes. Capacity to predict fetal RHD genotype for maternal carriers of hybrid or pseudogene RHD variants requires stringent control of cfDNA levels. We conclude that fetal RHD genotyping is feasible in the Australian environment to avoid unnecessary anti-D immunoglobulin prophylaxis.

Impact of Preanalytical Variations in Blood-Derived Biospecimens on Omics Studies: Toward Precision Biobanking?

Research data and outcomes do vary across populations and persons, but this is not always due to experimental or true biological variation. Preanalytical components of experiments, be they biospecimen acquisition, preparation, storage, or transportation to the laboratory, may all contribute to apparent variability in research data, outcomes, and interpretation. The present review article and biobanking innovation analysis offer new insights with a summary of such preanalytical variables, for example, the type of blood collection tube, centrifugation conditions, long-term sample storage temperature, and duration, on output of omics analyses of blood-derived biospecimens: whole blood, serum, plasma, buffy coat, and peripheral blood mononuclear cells. Furthermore, we draw parallels from the field of precision medicine in this study, with a view to the future of "precision biobanking" wherein such preanalytical variations are carefully taken into consideration so as to minimize their influence on outcomes of omics data, analyses, and sensemaking, particularly in clinical omics applications. We underscore the need for using broadly framed, critical, independent, social and political science, and humanities research so as to understand the multiple possible future trajectories of, and the motivations and values embedded in, precision biobanking that is increasingly relevant in the current age of Big Data.

A user's guide for α-synuclein biomarker studies in biological fluids: Perianalytical considerations.

Parkinson's disease biomarkers are needed to increase diagnostic accuracy, to objectively monitor disease progression and to assess therapeutic efficacy as well as target engagement when evaluating novel drug and therapeutic strategies. This article summarizes perianalytical considerations for biomarker studies (based on immunoassays) in Parkinson's disease, with emphasis on quantifying total α‐synuclein protein in biological fluids. Current knowledge and pitfalls are discussed, and selected perianalytical variables are presented systematically, including different temperature of sample collection and types of collection tubes, gradient sampling, the addition of detergent, aliquot volume, the freezing time, and the different thawing methods. We also discuss analytical confounders. We identify gaps in the knowledge and delineate specific areas that require further investigation, such as the need to identify posttranslational modifications of α‐synuclein and antibody‐independent reference methods for quantification, as well as the analysis of potential confounders, such as comorbidities, medication, and phenotypes of Parkinson's disease in larger cohorts. This review could be used as a guideline for future Parkinson's disease biomarker studies and will require regular updating as more information arises in this growing field, including new technical developments as they become available. In addition to reviewing best practices, we also identify the current technical limitations and gaps in the knowledge that should be addressed to enable accurate and quantitative assessment of α‐synuclein levels in the clinical setting. © 2017 The Authors. Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder Society.

Effects of sample processing and storage on the integrity of cell-free miRNAs in maternal plasma.

Cell-free fetal miRNAs have been identified as potential biomarkers for fetal abnormalities and/or placental function. Factors affecting the stability of cell-free fetal miRNA samples (type of collection tube and time interval between sampling and analysis) have not previously been reported. Blood from pregnant women (n=12, 18±4weeks' gestation) was collected into two types of tube (EDTA and RNA BCT) and was stored at different temperatures for up to 72h. Expression of seven apparently placental specific miRNAs was then measured to compare the effects of sampling and storage. These miRNAs were also assessed in non-pregnant women (n=9). The quantity of miRNA extracted was not affected by time or tube. Three miRNAs (miR-518b, miR-525 and miR-526a*) were measureable only in pregnant women, but miR-518b was not always present. Detailed study of the two pregnancy specific miRNAs showed no effect of tube type at 4h. However, variability in miRNA level was observed with increased time and was significant for one miRNA in the BCT tube at >48h (p<0.005). Some cffmiRNAs are placental specific, and these samples are stable when analyzed within 48h of collection in either tube type. © 2017 John Wiley & Sons, Ltd.

Abstract 5683: Evaluation of the utilization of blood collection tubes for cell-free DNA research

Abstract Many studies have been conducted investigating cell-free DNA (cfDNA) for disease diagnosis. To prevent cellular DNA release into the plasma, plasma separation within 2 hours of blood collection is required, when utilizing the most commonly used type of tube (BD Vacutainer® CPTTM; CPT). These temporal restrictions render operations requiring plasma separation a laborious task. It has been demonstrated that Cell-Free DNA BCT® tubes (Streck Inc; BCTs) prevented nucleated cell disruption. The use of BCTs may enable us to separate plasma without temporal restrictions. The aim of this study was to evaluate changes in DNA levels in plasma after preservation using two types of collection tubes. Samples were drawn from 7 healthy donors into CPTs and BCTs, and stored at room temperature. Plasma was separated immediately (d0), and on days 3, 6, 9, 12, and 14 after blood collection. The concentrations of DNA in the extracted plasma samples were measured. Blood stored in CPTs showed increases in plasma DNA concentrations over time. The mean DNA concentrations at each time-point were 0.39 (d0), 2.96 (d3), 40.4 (d6), 84.9 (d9), 91.4 (d12), and 94.9 ng/μl (d14). However, plasma DNA levels remained stable in BCTs from 6 days. There were significant differences between the DNA concentrations in BCTs on d0 (0.74 [0.43-1.44] ng/µl) and d14 (1.78 [0.63-4.20] ng/µl) (p = 0.0378). Although significant differences were only observed between d0 and d14 in BCTs, the amount of plasma DNA gradually increased after d6. Furthermore, obvious color changes in the plasma were observed, and the boundary between plasma and PBMCs became unclear over time. These results indicate that BCTs may yield stable cfDNA samples for up to 6 days after blood collection. BCTs may be a novel collection tube for cfDNA research, including liquid biopsies in cancer patients. Citation Format: Fumitaka Endo, Takeshi Iwaya, Takehiro Chiba, Mizunori Yaegashi, Kei Sato, Kohei Kume, Atsuhiro Arisue, Yutaka Nishinari, Ryoko Kawagishi, Takenori Segawa, Satoshi Nishizuka, Akira Sasaki. Evaluation of the utilization of blood collection tubes for cell-free DNA research [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5683. doi:10.1158/1538-7445.AM2017-5683

The relationship between vacuum and hemolysis during catheter blood collection: a retrospective analysis of six large cohorts.

Blood collection through intravenous (IV) catheters is a common practice at emergency departments (EDs). This technique is associated with higher in vitro hemolysis rates and may even be amplified by the use of vacuum collection tubes. Our aim was to investigate the association of five different vacuum tubes with hemolysis rates in comparison to an aspiration system under real-life conditions and to propose an equation to estimate the amount of hemolysis, depending on the vacuum collection tube type. We retrospectively evaluated hemolysis data of plasma samples from our ED, where blood is drawn through IV catheters. Over the past 5 years, we compared 19,001 hemolysis index values amongst each other and against the respective vacuum pressure (Pv) of the collection tubes, which were used within the six observational periods. The highest hemolysis rates were associated with full-draw evacuated tubes. Significantly reduced hemolysis was observed for two kinds of partial-draw tubes. The hemolysis rate of one partial-draw blood collection tube was comparable to those of the aspiration system. Regression analysis of Pv and mean free hemoglobin (fHb) values yielded the formula fHb (g/L)=0.0082*Pv2-0.1143*Pv+ 0.5314 with an R2 of 0.99. If IV catheters are used for blood collection, hemolysis rates directly correlate with the vacuum within the tubes and can be estimated by the proposed formula. By the use of partial-draw vacuum blood collection tubes, hemolysis rates in IV catheter collections can be reduced to levels comparable with collections performed by aspiration systems.

Effect of collection tube type and freeze-thaw cycles on myeloperoxidase concentrations in blood samples of acute coronary syndrome patients.

Background Myeloperoxidase has shown potential as a marker for prognosis of coronary artery disease, but presently little is known about preanalytical handling of samples for quantifying myeloperoxidase. The present study was conducted to evaluate the effect of collection tube and freeze-thaw cycles on myeloperoxidase concentrations. Methods Acute coronary syndrome patients ( n = 88) were enrolled after obtaining written informed consent from coronary care unit of a tertiary care hospital (January 2012-June 2014). About 5 mL venous blood was collected from patients and divided into serum, lithium heparin, ethylenediaminetetraacetic acid and sodium citrate tubes. Except serum, all tubes were kept on ice immediately after collection. Samples were centrifuged at -4℃, separated immediately after centrifugation and stored at -40℃ until analysis. Myeloperoxidase was quantified by in-house and commercial assays and re-quantified after five freeze-thaw cycles. Results Myeloperoxidase concentrations, (serum samples) determined by commercial and in-house assays correlated well (r = 0.946) ( P < 0.001) and were higher in serum samples. Within plasma, myeloperoxidase concentrations were slightly higher in ethylenediaminetetraacetic acid (307.7 ± 52.1) and lower in lithium heparin (290.3 ± 49.2) and sodium citrate (221.4 ± 40.3) but not statistically significant. Correlation between myeloperoxidase concentrations (in-house enzyme-linked immunosorbent assay) after first cycle and fifth freeze-thaw cycle dropped to r = 0.448 ( P < 0.001). Conclusion Myeloperoxidase concentrations are comparable in three types of plasma tubes when samples are placed on ice immediately, centrifuged at low temperatures and separated immediately after centrifugation. Multiple freeze-thaw cycles have an effect on myeloperoxidase and should be avoided for quantifying myeloperoxidase.

Identification of Analytical Factors Affecting Complex Proteomics Profiles Acquired in a Factorial Design Study with Analysis of Variance: Simultaneous Component Analysis.

Complex shotgun proteomics peptide profiles obtained in quantitative differential protein expression studies, such as in biomarker discovery, may be affected by multiple experimental factors. These preanalytical factors may affect the measured protein abundances which in turn influence the outcome of the associated statistical analysis and validation. It is therefore important to determine which factors influence the abundance of peptides in a complex proteomics experiment and to identify those peptides that are most influenced by these factors. In the current study we analyzed depleted human serum samples to evaluate experimental factors that may influence the resulting peptide profile such as the residence time in the autosampler at 4 °C, stopping or not stopping the trypsin digestion with acid, the type of blood collection tube, different hemolysis levels, differences in clotting times, the number of freeze-thaw cycles, and different trypsin/protein ratios. To this end we used a two-level fractional factorial design of resolution IV (2(IV)(7-3)). The design required analysis of 16 samples in which the main effects were not confounded by two-factor interactions. Data preprocessing using the Threshold Avoiding Proteomics Pipeline (Suits, F.; Hoekman, B.; Rosenling, T.; Bischoff, R.; Horvatovich, P. Anal. Chem. 2011, 83, 7786-7794, ref 1) produced a data-matrix containing quantitative information on 2,559 peaks. The intensity of the peaks was log-transformed, and peaks having intensities of a low t-test significance (p-value > 0.05) and a low absolute fold ratio (<2) between the two levels of each factor were removed. The remaining peaks were subjected to analysis of variance (ANOVA)-simultaneous component analysis (ASCA). Permutation tests were used to identify which of the preanalytical factors influenced the abundance of the measured peptides most significantly. The most important preanalytical factors affecting peptide intensity were (1) the hemolysis level, (2) stopping trypsin digestion with acid, and (3) the trypsin/protein ratio. This provides guidelines for the experimentalist to keep the ratio of trypsin/protein constant and to control the trypsin reaction by stopping it with acid at an accurately set pH. The hemolysis level cannot be controlled tightly as it depends on the status of a patient's blood (e.g., red blood cells are more fragile in patients undergoing chemotherapy) and the care with which blood was sampled (e.g., by avoiding shear stress). However, its level can be determined with a simple UV spectrophotometric measurement and samples with extreme levels or the peaks affected by hemolysis can be discarded from further analysis. The loadings of the ASCA model led to peptide peaks that were most affected by a given factor, for example, to hemoglobin-derived peptides in the case of the hemolysis level. Peak intensity differences for these peptides were assessed by means of extracted ion chromatograms confirming the results of the ASCA model.

The Influence of Blood Collection Tubes on Measurement of Cardiac Biomarkers.

The tube used to collect a blood specimen may have an effect on some test results. This study evaluated three different types of blood collection tubes manufactured in China for use in testing three cardiac biomarkers. Blood samples were drawn from 42 patients in the Intensive Care Unit and were sampled into three different types of blood collection tubes at the same time. All samples were subjected to analysis of myoglobin (MYO), creatine kinase MB (CK-MB), and high-sensitive cardiac troponin T (hs-cTnT) using the Roche Cobas e411 chemistry analyzer. There was no statistically significant difference in the test results for MYO and CK-MB among the three different types of blood collection tubes. However, there was a statistically significant difference in the measured level of hs-cTnT. The hs-cTnT values in tubes with clot activator were significantly lower than the values from tubes with no additive (p = 0.000) or lithium heparin (p = 0.002). Three types of blood collection tubes are safe for myoglobin and CK-MB determination without altering the results. However, the hs-cTnT value was lower in clot activator tubes than in tubes with no additive or with heparin lithium. Thus, we conclude that using clot activator tubes can affect the determination of hs-cTnT concentration which should be noted in clinical practice.

Examining factors that may influence accurate measurement of testosterone in sea turtles.

Differences in reported testosterone concentrations in male sea turtle blood samples are common in the veterinary literature, but may be accounted for by differences in sample handling and processing prior to assay. Therefore, our study was performed to determine best practices for testosterone analysis in male sea turtles (Caretta caretta and Chelonia mydas). Blood samples were collected into 5 collection tube types, and assay validation and measured testosterone concentrations were compared across different sample storage (fresh, refrigerated 1 week, or frozen), extraction (unextracted or ether-extracted), and processing treatment (untreated, homogenized, or dissociation reagent) conditions. Ether-extracted and dissociation reagent-treated samples validated in all conditions tested and are recommended for use, as unextracted samples validated only if assayed fresh. Dissociation reagent treatment was simpler to perform than ether extraction and resulted in total testosterone concentrations ~2.7-3.5 times greater than free testosterone measured in ether-extracted samples. Sample homogenization did not affect measured testosterone concentrations, and could be used to increase volume in gelled samples. An annual seasonal testosterone increase was observed in both species when ether extraction or dissociation reagent treatment was used. Annual deslorelin implant treatments in a Chelonia mydas male resulted in suppression of seasonal testosterone following the fourth treatment. Seasonal testosterone patterns resumed following discontinuation of deslorelin. Comparison of in-house and commercially available enzyme immunoassay kits revealed similar patterns of seasonal testosterone increases and deslorelin-induced suppression. Our study highlights the importance of methodological validation and provides laboratorians with best practices for testosterone enzyme immunoassay in sea turtles.

In-vitro hemolysis and its financial impact using different blood collection systems

Abstract Background: Hemolytic specimens are among the most challenging preanalytical issues in laboratory diagnostics. The type of blood collection tube in use is claimed to influence in vitro hemolysis. We aimed to examine this hypothesis and estimate the respective financial impact, evaluating routine blood samples from the past 4 years. Methods: A total of 47,820 hemolysis index (HI) values from five different time intervals (IV1–IV5) were compared against each other, representing the following tubes: IV1-Sarstedt Monovette; IV2-8 mL/16×100 mm Greiner BioOne (GBO) Vacuette; IV3/IV4-5 mL/16×100 mm GBO Vacuette; IV5-4.5 mL/13×75 mm GBO Vacuette. For estimation of the economic impact, material, personnel and analytical costs were calculated. Results: HI mean values in time interval IV2 were significantly higher than in all other intervals, while mean values amongst all other intervals were comparable. The number of moderately and severely hemolyzed samples increased with incrementing vacuum. Overall comparable costs between intervals IV1 and IV5 were €11,370, €14,045, €12,710, €11,213 and €8138 per 10,000 samples, respectively. Conclusions: Aspiration tubes and low vacuum tubes show comparable hemolysis rates. Increasing vacuum levels are associated with higher hemolysis rates. By decreasing in vitro hemolysis, financial savings up to €5907 per 10,000 samples could be gained.

LC-MS/MS Method for Determination of Teriflunomide, Over a 40,000-Fold Dynamic Range Using Overlapping Calibrators.

A liquid chromatography tandem mass spectrometry (LC-MS/MS) method has been validated for use in therapeutic monitoring of the drug leflunomide in human serum and plasma. Because of concerns of teratogenicity, it is recommended that women who want to become pregnant have concentrations below 0.02 mcg/mL, although therapeutic levels are generally greater than 20 mcg/mL. Consequently, the method required a 40,000-fold dynamic range, which was achieved by dividing the curve range into 2 separate regions but with a single extraction procedure used for both. A chromatographic separation was achieved between the parent drug and the active metabolite, teriflunomide (A77 1726), and the latter was quantified across a quantitative range of 0.005-200 mcg/mL. Samples were evaluated in an upper curve region first, with dilution, to determine whether the drug concentrations were in an appropriate therapeutic range. Samples that fell below the upper region were then reevaluated in the lower region without dilution. The method was shown to be reliable, with good accuracy and precision statistics, and acceptable quantitation using 4 different collection tube types. Mean accuracy over 6 control concentrations was within 5.4%, over 5 validation runs, whereas %coefficient of variation (CV) was within 8.15%. Evaluation of sodium heparin, KEDTA, NaF/K oxalate, and plain serum tubes from 6 separate individuals at the lower limit of quantification (LLOQ) showed no influence on the ability to quantify teriflunomide accurately. Regression equations for a curve range of 0.005-1 mcg/mL gave R values of 0.998 or better, whereas the range 0.8-200 mcg/mL had R values of 0.997 or better. The authors have developed and validated a method that allows quantification of leflunomide across a 40,000-fold range of 0.005-200 mcg/mL.

Real-time PCR evaluation of cell-free DNA subjected to various storage and shipping conditions.

In this study, we attempted to explore the factors affecting the yield of cell-free fetal DNA (cffDNA) obtained from maternal blood samples, including the use of different types of collection tubes, the interval between sample processing, and sample shipping under extreme weather conditions. Blood samples were drawn into K3EDTA tubes and cell-stabilizing tubes (Streck blood collection tube, BCT) from women pregnant with male fetuses. Real time PCR was used to amplify a β-actin gene fragment to measure the total plasma cell-free DNA concentration, while an SRY gene fragment was used to quantify the cffDNA. The samples in the K3EDTA tubes revealed a decreased quantity of SRY after 5 days of transportation, with a median of 25.9 copies/mL (P < 0.01); however, the value remained stable at 33.4 copies/mL in the BCT tubes. We observed a statistically significant increase in stability of the amount of total DNA in the blood samples stored in K3EDTA tubes (P < 0.01) and transportated under extreme outdoor temperatures (-20°-0°C) than that of the control values. These results indicate that it could be possible to avoid the presence of excess maternal DNA in samples shipped under extreme weather conditions for no more than 2 days, by collecting the blood samples in BCT tubes.

Detection of sCD21 in Human Blood Using ELISA is Influenced by Ethylenediaminetetraacetic Acid (EDTA) Containing Blood Collection Tubes.

CD21 is a 145 kDa membrane glycoprotein expressed mainly on B-cells and follicular dendritic cells. It is involved in activation, survival and proliferation of B-cells. CD21 can be cleaved to give soluble CD21 (sCD21), which is constantly shed in healthy people whereas the level of sCD21 is low in patients suffering from various autoimmune diseases. Blood was collected from 12 healthy donors into Heparin, EDTA and gel-separation tubes. Furthermore, plasma from 6 healthy donors was combined and EDTA or EGTA was subsequently added to analyze its influence on sCD21 levels measured by ELISA. Differences in sCD21 levels were observed dependent on blood collection tubes used. sCD21 levels measured by ELISA were significantly higher if heparin/gel containing tubes were used compared to EDTA blood collection tubes. Upon addition of EDTA/EGTA to plasma drawn using Heparin blood collection tubes, sCD21 levels decrease to amounts found in EDTA-containing blood collection tubes suggesting calcium as the responsible factor rather than magnesium. EDTA influences sCD21 levels determined by ELISA and therefore sCD21 measurements should be carried out using one type of blood collection tube throughout the experiment/medical examination.

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

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.

Biomarkers for monitoring pre-analytical quality variation of mRNA in blood samples.

There is an increasing need for proper quality control tools in the pre-analytical phase of the molecular diagnostic workflow. The aim of the present study was to identify biomarkers for monitoring pre-analytical mRNA quality variations in two different types of blood collection tubes, K2EDTA (EDTA) tubes and PAXgene Blood RNA Tubes (PAXgene tubes). These tubes are extensively used both in the diagnostic setting as well as for research biobank samples. Blood specimens collected in the two different blood collection tubes were stored for varying times at different temperatures, and microarray analysis was performed on resultant extracted RNA. A large set of potential mRNA quality biomarkers for monitoring post-phlebotomy gene expression changes and mRNA degradation in blood was identified. qPCR assays for the potential biomarkers and a set of relevant reference genes were generated and used to pre-validate a sub-set of the selected biomarkers. The assay precision of the potential qPCR based biomarkers was determined, and a final validation of the selected quality biomarkers using the developed qPCR assays and blood samples from 60 healthy additional subjects was performed. In total, four mRNA quality biomarkers (USP32, LMNA, FOSB, TNRFSF10C) were successfully validated. We suggest here the use of these blood mRNA quality biomarkers for validating an experimental pre-analytical workflow. These biomarkers were further evaluated in the 2nd ring trial of the SPIDIA-RNA Program which demonstrated that these biomarkers can be used as quality control tools for mRNA analyses from blood samples.

Pilot study to assess effects of collection tube types and processing delay on measurements of persistent organic pollutants and lipids in human serum

Glass red top tubes (RTs), traditionally used to draw blood for biomonitoring studies, have some limitations during field sampling (e.g., tube breakage, timely processing may be difficult). This pilot study examined whether serum separation tubes (SSTs) with delayed processing time (48h) can be used instead of red top tubes (RTs) to accommodate field conditions. Using state-of-the-art methodologies, PBDEs, PCBs, OCPs, PFCs, cholesterol and triglycerides were measured to evaluate any differences among 2 test conditions (RTs with 2h processing time; SSTs with 48h processing time). Between the 2 test conditions, we observed high rank correlations among the measured compounds and no statistically significant differences in the levels of measured compounds. We conclude that SSTs with delayed processing time (48h) produce similar results as RTs with short processing time (2h), suggesting that SSTs could be good substitutes for RTs for new epidemiological and biomonitoring field studies. The use of SSTs offers a tremendous opportunity for the use of samples archived in various SSTs.

Biomarkers for neurological disorders – call for standardization

Abstract With increasing life expectancy, neurodegenerative diseases such as Alzheimer’s disease (AD) and Parkinson’s disease (PD), as well as neurovascular diseases such as cerebral ischemia, are becoming a rapidly growing worldwide problem causing an increased burden on society. The development of biomarkers is aimed to enable the identification of at-risk individuals, enable early diagnosis, and reflect the progression of disease. Future challenges refer to the need for standardization of known biomarkers on the one hand and the identification of new biomarkers on the other hand. The former is impressively exemplified by a recent study addressing the impact of various types of cerebrospinal fluid collection tubes for detection of total tau, phosphorylated tau, and β-amyloid 1–42 on the diagnosis of AD. This issue was identified as a key issue by the Alzheimer’s Biomarkers Standardization Initiative (ABSI) and recommendations were listed which should be used to enable better standardization between laboratories and enable results for the AD biomarkers to be comparable between different sites. The latter challenge in particular concerns PD and is addressed by the Parkinson Progression Marker Initiative (PPMI), a comprehensive observational, international, multi-center study designed to identify PD progression biomarkers both to improve understanding of disease etiology and course and to provide crucial tools to enhance the likelihood of success of PD modifying therapeutic trials. With regard to cerebral ischemia, at present there are no clinically validated biomarkers. Nevertheless, the development of a clinically validated biomarker of acute cerebral ischemia would have the potential to facilitate the use of time-sensitive reperfusion strategies, allow for individualization of patient care by predicting relative risk of hemorrhage and volume of penumbral tissue, and add valuable prognostic information for patients presenting with acute stroke. Thereby, it is expected that a panel of biomarkers rather than one single marker will meet these criteria in the future. Unlike cerebral ischemia, cerebral hemorrhage biomarkers will probably be limited to the assessment of prognosis in the course of the disease.