Types Of Blood Collection Tubes Research Articles

Cell-Free Microbial DNA Analysis: Effects of Blood Plasma and Serum Quantity, Biobanking Protocols, and Isolation Kits.

Recent studies highlight the presence of bacterial sequences in the human blood, suggesting potential clinical significance for circulating microbial signatures. These sequences could presumably serve in the diagnosis, prediction, or monitoring of various health conditions. Ensuring the similarity of samples before bacterial analysis is crucial, especially when combining samples from different biobanks prepared under varying conditions (such as different DNA extraction kits, centrifugation conditions, blood collection tubes, etc.). In this study, we aimed to analyze the impact of different sample collection and nucleic acid extraction criteria (blood collection tube, centrifugation, input volume, and DNA extraction kit) on circulating bacterial composition. Blood samples from four healthy individuals were collected into three different sample collection tubes: K2EDTA plasma tube, sodium citrate plasma tube, and gel tube for blood serum. Tubes were centrifugated at standard and double centrifugation conditions. DNA extraction was performed using 100, 200, and 500 μL plasma/serum input volumes. DNA extraction was performed using three different isolation kits: Norgen plasma/serum cell-free circulating DNA purification micro kit, Applied Biosystems MagMAX cell-free DNA isolation kit, and Qiagen QIAamp MinElute cell-free circulating DNA mini kit. All samples were subjected to 16S rRNA V1-V2 library preparation and sequencing. In total, 216 DNA and 18 water control samples were included in the study. According to PERMANOVA, PCoA, Mann-Whitney, and FDR tests the effect of the DNA extraction kit on the microbiota composition was the greatest, whereas the type of blood collection tube, centrifugation type, and sample input volume for the extraction had minor effects. Samples extracted with the Norgen DNA extraction kit were enriched with Gram-negative bacteria, whereas samples extracted with the Qiagen and MagMAX kits were enriched with Gram-positive bacteria. Bacterial profiles of samples prepared with the Qiagen and MagMAX DNA extraction kits were more similar, whereas samples prepared with the Norgen DNA extraction kit were significantly different from other groups.

Electrochemical analysis of antioxidant status of biological media in different sampling and storage conditions

The use of an electrochemical approach for assessment of the oxid-ative stress severity is a promising direction for point-of-care testing develop-ment, which is especially important for critically ill patients. The aim of this study was to determine the influence of different types of blood collection tubes (with clot activator and separating gel, lithium heparin, sodium citrate and K2EDTA) and storage conditions (at 25, 4 and ?23 ?C up to 5 days) on the electrochemical analysis of the antioxidant status of blood plasma, assessed by measuring the open circuit potential of platinum electrode (OCP) and antioxid-ant capacity via cyclic voltammetry method (q). It was obtained that blood col-lection tubes with lithium heparin and clot activator are the most suitable for the electrochemical analysis of antioxidant status of blood plasma, since they do not affect the results of measurements. Furthermore, data obtained during storage blood plasma samples in different temperature conditions indicate that it is preferable to perform electrochemical analysis in fresh samples.

Evaluation of Small Molecules in Blank EDTA Plasma Tubes and Optimization of Metabolomic Workflow for Biomarker Studies Using Plasma Samples.

As the first step of metabolomic analysis in biomarker identification studies, various types of blood collection tubes are used in clinical practice. However, little attention is paid to potential contamination caused by the blank tube itself. Here, we evaluated small molecules in blank EDTA plasma tubes through LC-MS-based untargeted metabolomic analysis and identified small molecules with markedly varied levels among different production batches or specifications. Our data demonstrate possible contamination and data interference caused by blank EDTA plasma tubes when employing large clinical cohorts for biomarker identification. Therefore, we propose a workflow of filtering metabolites in blank tubes prior to statistical analysis to improve the fidelity of biomarker identification.

Effects of sample matrix in the measurement of antithrombin by LC-MS: A role for immunocapture

IntroductionThe sample matrix composition, which is greatly affected by the type of blood collection tube used during phlebotomy, is of major importance in laboratory testing as it can influence test results. We developed an LC-MRM-MS test to molecularly characterize antithrombin in citrate plasma. The test principle differs greatly from traditional laboratory tests and the influence of varying plasma sample matrices is largely unknown. ObjectivesTo identify whether variations in sample matrix affect the LC-MRM-MS test for antithrombin and assess whether sample pre-processing by immunocapture reduces matrix-specific effects. MethodsSamples (n = 45) originating from four different blood collection tubes (sodium citrate, lithium heparin, K2-EDTA and K2-EDTA with protease inhibitors) were processed directly or after immunocapture. Antithrombin was digested into proteotypic peptides, which were monitored by LC-MRM-MS. Results from lithium heparin and the K2-EDTA matrices were compared to the standard sample matrix, sodium citrate, using Deming regression analysis and repeated measures one-way ANOVA. ResultsDeming regression analysis of directly processed samples revealed slopes deviating >5% from the line of identity for at least six out of 22 peptides in all matrices. Significant differences between all matrices were found upon analysis by ANOVA for at least 10 peptides. Pre-processing by immunocapture led to slopes within 5% of the line of identity for nearly all peptides of the matrices. Furthermore, significant differences between matrices after immunocapture were only observed for four peptides. ConclusionVariations in the sample matrix affect the measurement of antithrombin by LC-MRM-MS, but observed effects are greatly reduced upon pre-processing by immunocapture.

The Stability of Intact Parathyroid Hormone (PTH) in Different Types of Blood Collection Tubes.

Over past decades, the instability of parathyroid hormone (PTH) causes great interference for the clinical laboratory. Contradictory results were reported in many reports about storage conditions and suitable blood collection tubes to ensure PTH stability in the pretreatment phase. This study recruited 30 participants including 10 healthy persons, 10 hemodialysis, and 10 hyperparathyroidism patients. Five types of blood collection tubes (EDTA-K3 tube, coagulant tube, heparin anticoagulant tube, gel separating tube, and plain tube) were included to determine whether they were suitable as blood-collecting vessels. The time points and conditions for testing samples included less than 2 hours, 4 hours, and 8 hours at room temperature, and, in parallel, 24 hours, 48 hours, and 72 hours in refrigeration. Two different judgement criteria were used to compare the stability of PTH in different blood vessels. Purely statistical analysis showed that 4 types of blood collection tubes could not perform the same storage ability as EDTA-K3 tube at "T0" time point. Plain tube had the largest drop among all types of blood collection tubes. Compared by pairwise t-test, EDTA-K3 tube could maintain intact PTH for 8 hours (p = 0.998) at room temperature and 24 hours (p = 0.053) in refrigeration. When comparing the total change limit (TCL = 18.8%), at room temperature, EDTA-K3 tube (7.0%), heparin tube (12.7%), coagulant tube (16.2%), and plain tube (17.6%) could maintain intact PTH for 8 hours, and GST can preserve PTH for 4 hours (18.2%). In refrigeration, EDTA-K3 tube could maintain PTH for 72 hours (7.5%) and heparin tube could maintain 24 hours (18.4%). The other three blood collection tubes could not preserve PTH in refrigeration (GST = 22.1%, coagulant tube = 20.3%, plain tube = 20.8%). PTH seems more stable in the EDTA-K3 tube than any other blood collection tubes and is followed next by the heparin anticoagulant tube. Plain tube and GST have faster degradation than other tubes and are not suggested to preserve intact PTH.

Evaluation of BD Barricor™ and PST™ blood collection tubes compared to serum for testing 11 therapeutic drugs on a Roche Cobas® 8000 platform

IntroductionThe type of blood collection tube used when obtaining samples for therapeutic drug monitoring (TDM) has important implications on the accuracy of results. Serum tubes without a gel separator are currently considered best practice. We sought to evaluate the performance of Barricor™, a novel plasma tube that utilizes an inert mechanical separator, as well as a gel-based tube (PST™) for testing acetaminophen, digoxin, gentamicin, methotrexate, phenobarbital, phenytoin, salicylate, vancomycin, valproic acid, carbamazepine, and theophylline on a Roche Cobas® 8000 platform. MethodsPaired patient samples were collected from individuals taking at least one of the medications evaluated. These were supplemented with spiked specimens to ensure a minimum of 40 paired samples per drug. All drugs were measured within two hours of collection on Roche e602 or c502 instruments. Deming regression was used to assess bias between Barricor™ vs serum and PST™ vs serum. Seven-day refrigerated stability was also assessed in Barricor™, PST™, and serum tubes in a subset of samples (n = 10) for each drug. ResultsDrug concentrations in Barricor™ were similar to serum for each drug assessed. In contrast, a negative bias was observed in PST™ compared to serum tubes for carbamazepine (−7.6%) and phenytoin (−6.8%) although this did not surpass our total allowable error goal of 10%. All drugs recovered within ±10% of baseline value when samples were stored refrigerated for 7 days except for carbamazepine, phenytoin, and phenobarbital where significant analyte loss was observed within the first day in PST™ tubes. ConclusionBarricor™ tubes are a suitable alternative to serum for TDM on the Roche Cobas® 8000 platform.

High-throughput and affordable genome-wide methylation profiling of circulating cell-free DNA by methylated DNA sequencing (MeD-seq) of LpnPI digested fragments

BackgroundDNA methylation detection in liquid biopsies provides a highly promising and much needed means for real-time monitoring of disease load in advanced cancer patient care. Compared to the often-used somatic mutations, tissue- and cancer-type specific epigenetic marks affect a larger part of the cancer genome and generally have a high penetrance throughout the tumour. Here, we describe the successful application of the recently described MeD-seq assay for genome-wide DNA methylation profiling on cell-free DNA (cfDNA). The compatibility of the MeD-seq assay with different types of blood collection tubes, cfDNA input amounts, cfDNA isolation methods, and vacuum concentration of samples was evaluated using plasma from both metastatic cancer patients and healthy blood donors (HBDs). To investigate the potential value of cfDNA methylation profiling for tumour load monitoring, we profiled paired samples from 8 patients with resectable colorectal liver metastases (CRLM) before and after surgery.ResultsThe MeD-seq assay worked on plasma-derived cfDNA from both EDTA and CellSave blood collection tubes when at least 10 ng of cfDNA was used. From the 3 evaluated cfDNA isolation methods, both the manual QIAamp Circulating Nucleic Acid Kit (Qiagen) and the semi-automated Maxwell® RSC ccfDNA Plasma Kit (Promega) were compatible with MeD-seq analysis, whereas the QiaSymphony DSP Circulating DNA Kit (Qiagen) yielded significantly fewer reads when compared to the QIAamp kit (p < 0.001). Vacuum concentration of samples before MeD-seq analysis was possible with samples in AVE buffer (QIAamp) or water, but yielded inconsistent results for samples in EDTA-containing Maxwell buffer. Principal component analysis showed that pre-surgical samples from CRLM patients were very distinct from HBDs, whereas post-surgical samples were more similar. Several described methylation markers for colorectal cancer monitoring in liquid biopsies showed differential methylation between pre-surgical CRLM samples and HBDs in our data, supporting the validity of our approach. Results for MSC, ITGA4, GRIA4, and EYA4 were validated by quantitative methylation specific PCR.ConclusionsThe MeD-seq assay provides a promising new method for cfDNA methylation profiling. Potential future applications of the assay include marker discovery specifically for liquid biopsy analysis as well as direct use as a disease load monitoring tool in advanced cancer patients.

Comparative metabolomics studies of blood collected in streck and heparin tubes from lung cancer patients.

Metabolomics analysis of blood from patients (n = 42) undergoing surgery for suspected lung cancer was performed in this study. Venous and arterial blood was collected in both Streck and Heparin tubes. A total of 96 metabolites were detected, affected by sex (n = 56), collection tube (n = 33), and blood location (n = 8). These metabolites belonged to a wide array of compound classes including lipids, acids, pharmaceutical agents, signalling molecules, vitamins, among others. Phospholipids and carboxylic acids accounted for 28% of all detected compounds. Out of the 33 compounds significantly affected by collection tube, 18 compounds were higher in the Streck tubes, including allantoin and ketoleucine, and 15 were higher in the Heparin tubes, including LysoPC(P-16:0), PS 40:6, and chenodeoxycholic acid glycine conjugate. Based on our results, it is recommended that replicate blood samples from each patient should be collected in different types of blood collection tubes for a broader range of the metabolome. Several metabolites were found at higher concentrations in cancer patients such as lactic acid in Squamous Cell Carcinoma, and lysoPCs in Adenocarcinoma and Acinar Cell Carcinoma, which may be used to detect early onset and/or to monitor the progress of the cancer patients.

Genome-wide study of the effect of blood collection tubes on the cell-free DNA methylome

ABSTRACT The methylation pattern of cfDNA, isolated from liquid biopsies, is gaining substantial interest for diagnosis and monitoring of diseases. We have evaluated the impact of type of blood collection tube and time delay between blood draw and plasma preparation on bisulphite-based cfDNA methylation profiling. Fifteen tubes of blood were drawn from three healthy volunteer subjects (BD Vacutainer K2E EDTA spray tubes, Streck Cell-Free DNA BCT tubes, PAXgene Blood ccfDNA tubes, Roche Cell-Free DNA Collection tubes and Biomatrica LBgard blood tubes in triplicate). Samples were either immediately processed or stored at room temperature for 24 or 72 hours before plasma preparation. DNA fragment size was evaluated by capillary electrophoresis. Reduced representation bisulphite sequencing was performed on the cell-free DNA isolated from these plasma samples. We evaluated the impact of blood tube and time delay on several quality control metrics. All preservation tubes performed similar on the quality metrics that were evaluated. Furthermore, a considerable increase in cfDNA concentration and the fraction of it derived from NK cells was observed after a 72-hour time delay in EDTA tubes. The methylation pattern of cfDNA is robust and reproducible in between the different preservation tubes. EDTA tubes processed as soon as possible, preferably within 24 hours, are the most cost effective. If immediate processing is not possible, preservation tubes are valid alternatives.

Abstract B49: Substantial performance differences among RNA purification kits and blood collection tubes in the Extracellular RNA Quality Control study—important considerations for liquid biopsies

Abstract Cancer biomarker studies require procedures that provide accurate and precise test results with high analytical sensitivity. Consequently, the growing use of extracellular RNA from human biofluids as clinically relevant biomarker requires the implementation of benchmarked methods for sample collection, processing, and profiling. While several small-scale studies have pointed at the impact of individual preanalytical factors, no comprehensive study has addressed the many preanalytical variables affecting downstream sequencing of blood-derived exRNAs. In the Extracellular RNA Quality Control study, we have systematically evaluated the type of blood collection tube (n=10, including 5 so-called preservation tubes), the time between blood draw and plasma preparation (n=3), different plasma types (n=3, i.e., platelet-free, -poor, and -rich plasma), and RNA purification methods using the supplier-specified minimum and maximum plasma input volumes (n=15). The impact of these preanalytical factors is assessed by deep transcriptome profiling of all small and messenger RNAs from healthy donors’ plasma, using TruSeq Small RNA sequencing and TruSeq RNA Exome sequencing, respectively. All experiments are conducted in triplicate (for a total of 270 transcriptomes) using 191 synthetic RNA spike-in molecules as processing controls over a relevant dynamic range. When comparing blood collection tubes, serum mRNA seems very similar to EDTA plasma mRNA, but serum-derived small RNAs are markedly different in biotype composition compared to their plasma counterparts. Furthermore, several plasma tubes with preservation reagents do not stabilize RNA very well, as is reflected by increasing RNA concentrations and number of detected genes over time. Also, their reproducibility is generally compromised. In addition, we demonstrate large differences in RNA purification kit performance in terms of reproducibility, sensitivity, and observed transcriptome complexity. Among others, we note a 50-fold difference in mRNA yield and a 5-fold difference in the number of detected mRNAs. We summarized the results in 12 performance parameters that enable an informed selection of the most optimal sample processing workflow. In conclusion, using a systematic approach, we put forward robust quality control metrics for exRNA quantification methods with validated SOPs for sample collection, processing, and profiling. Our results are crucially important for all future RNA-based liquid biopsy-guided precision oncology applications. Authors in random order; abstract submitted on behalf of the exRNAQC Consortium. Citation Format: Anneleen Decock, Jasper Anckaert, Celine Everaert, Justine Nuytens, Scott Kuersten, An Hendrix, Jo Vandesompele, Pieter Mestdagh, Bert Dhondt, Ruben Van Paemel, Kimberly Verniers, Gary Schroth, Carolina Fierro, Nurten Yigit, Kathleen Schoofs, Annelien Morlion, Jill Deleu, Eva Hulstaert, Francisco Avila Cobos, Nele Nijs, Eveline Vanden Eynde, Hetty Hilde Helsmoortel, Olivier De Wever, Annouck Philippron. Substantial performance differences among RNA purification kits and blood collection tubes in the Extracellular RNA Quality Control study—important considerations for liquid biopsies [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B49.

The Influence of Blood Collection Tubes in Biomarkers' Screening by Mass Spectrometry.

Mass spectrometry is one of the rapidly developing bio-analytical techniques in recent years, and it shows that the results of biomarkers' screening can be influenced by pre-analytical process. The selection of the blood collection tubes is one of the most significant steps of pre-analytical process which is often neglected by researchers. So, it is urgent to define the influence of blood collection tubes clearly in biomarkers' screening. Two types of blood collection tubes, non-additive tubes and coagulant activator tubes, are used to collect serum samples from patients and healthy controls. All samples are analyzed using matrix-assisted laser desorption ionization-time of flight mass spectrum in this study. The serum protein profile changes while using coagulant tubes whether for patients or healthy controls. It is found that the effect of coagulant on serum protein of patients is smaller than that of control group. There are 27 significantly different peaks between the control group and the control coagulant group. However, between patient group and patient coagulant group, only one differential peak is obtained. Coagulant changes the expression differences between patients and healthy controls, making the differences expand, shrink or reverse, and most of the polypeptides are small molecule, which will change the results of biomarker's screening. This research suggested that different types of blood collection tubes would influence the final laboratory results. So it's important for clinicians to choose the proper tubes to detect biomarkers and make correct diagnoses.

Comparison of the Profile and TSH Levels from Several Types of Blood Collection Tubes

Thyroid-Stimulating Hormone (TSH) is an important parameter in diagnosing thyroid disease which uses serumaccording to the World Health Organization's (WHO) recommendations. The use of plasma can help improve the TurnAround Time (TAT); however, the discrepancy with serum is unknown. A cross-sectional study using 89 blood samples wasperformed to compare TSH levels using serum tubes with clot activator (Tube I), plasma tubes with heparin (Tube II), andplasma tubes with heparin-gel separator (Tube III); and to overview of TSH levels according to gender and age. The medianof TSH levels in Tubes I, II, and III were 1.380 (0.032-7.420) μIU/mL, 1.380 (0.030-7.480) μIU/mL, and 1.360 (0.030-7.460)μIU/mL, respectively. There were no statistically significant differences in TSH levels of the three tubes. The median TSHlevels differences of Tubes II and III compared to the tube I were -0.9% (-7.2-2.2) and -1.7% (-8.0-1.6), respectively.Measurement bias observed in this study was following the specified desirable bias according to Ricos. The median TSHlevels of the male and female groups were 1.500 (0.032-4.250) μIU/mL and 1.345 (0.058-7.420) μIU/mL, respectively. MedianTSH levels of 31-40 years old age group and &gt;61 years old age group were 1.190 (0.609-3.240) μIU/mL and 1.730 (0.088-5.760) μIU/mL, respectively. Specimens from three tubes could be used to examine TSH levels. Measurement of TSH levelsshowed a higher median in the male and older group.

Serum Levels of Coll2-1, a Specific Biomarker of Cartilage Degradation, Are Not Affected by Sampling Conditions, Circadian Rhythm, and Seasonality

To assess intraindividual biological variability of serum cartilage specific biomarker Coll2-1 and define the best standardized conditions for blood sampling. Blood samples were taken from 116 subjects with knee osteoarthritis (OA) at a single time point (PRODIGE study) and from 15 healthy subjects under various conditions, including fasting condition, sampling time and season, blood treatment, and type of blood collection tube (COVAR study). Type II collagen-specific biomarker Coll2-1 was directly measured in serum using an immunoassay. There was no significant difference on Coll2-1 values between samples collected at any of the 5 sampling times or at any of the sampling days measured. None of the sampling parameters tested had a significant impact on Coll2-1 value (clotting time, clotting temperature and temperature of blood centrifugation, type of tube). On the contrary, differences were found in between subjects and between subjects with knee OA and healthy subjects. Coll2-1 measurement is not affected by sampling specific conditions, circadian rhythm or seasons but was found elevated in subject with knee OA indicating that Coll2-1 serum variation is not linked to the study environment, but to cartilage degradation in OA. Coll2-1 assay is sufficiently robust for use in OA clinical trials.

Striking Differences in Platelet Distribution between Advanced-Platelet-Rich Fibrin and Concentrated Growth Factors: Effects of Silica-Containing Plastic Tubes.

Compared with platelet-rich plasma, the preparation of platelet-rich fibrin (PRF) is simple and has not been overly modified. However, it was recently demonstrated that centrifugation conditions influence the composition of PRF and that silica microparticles from silica-coated plastic tubes can enter the PRF matrix. These factors may also modify platelet distribution. To examine these possibilities, we prepared PRF matrices using various types of blood-collection tubes (plain glass tubes and silica-containing plastic tubes) and different centrifugation speeds. The protocols of concentrated growth factors and advanced-PRF represented high- and low-speed centrifugation, respectively. Platelet distribution in the PRF matrix was examined immunohistochemically. Using low-speed centrifugation, platelets were distributed homogeneously within the PRF matrix regardless of tube types. In high-speed centrifugation, platelets were distributed mainly on one surface region of the PRF matrix in glass tubes, whereas in silica-coated tubes, platelet distribution was commonly more diffusive than in glass tubes. Therefore, both blood-collection tube types and centrifugal conditions appeared to influence platelet distribution in the PRF matrix. Platelets distributed in the deep regions of the PRF matrix may contribute to better growth factor retention and release. However, clinicians should be careful in using silica-coated tubes because their silica microparticles may be a health hazard.

Influence of sample collection tube method, anticoagulant-containing plasma versus serum, on influenza virus hemagglutination inhibition titer and microneutralization titer serological assays

BackgroundThe hemagglutination-inhibition (HAI) assay is a critical component for measurement of immunogenicity in influenza vaccine development. It is unknown if the results can be influenced by sample type and anticoagulants. The purpose of this study was to evaluate the influence of different sample collection methods, in particular different anticoagulants, and choice of plasma or serum, on influenza virus serological assays.MethodsBlood samples from thirty donors previously immunized against influenza viruses were collected using six different types of blood collection tubes, two of which collect serum and four of which contain various anticoagulants for collecting plasma. Serum: (1) serum separator tubes (SST); and (2) Plus Plastic serum “red-top serum” tubes. Plasma: (3) spray-coated K2 ethylenediaminetetraacetic acid (EDTA) tubes: (4) Sodium Heparin tubes; (5) Citrate tubes with 3.2% sodium citrate solution; and (6) Glass Blood Collection tubes with acid citrate dextrose. Samples were tested against three different influenza viruses (A/California/07/2009 (H1N1pdm09), A/Texas/50/2012 (H3N2), and B/Massachusetts/2/2012) for hemagglutination inhibition titer and virus neutralization titer via a microneutralization (MN) assay, and data compared to that obtained for standard serum sample collected in SST.ResultsHAI and MN titers against type A viruses were within two dilutions compared to SST collection method over 96% of the time irrespective of sample type or anticoagulant. However, HAI titers for type B virus were more variable across different collection methods. EDTA plasma samples were greater than two dilutions higher than SST serum samples 70% (21 of 30 samples) of the time. In contrast, MN titers were within two dilutions over 96% of the time, with the highest deviation noted in acid citrate dextrose plasma samples (3 of 30 samples tested, 10%).ConclusionsThese data provide useful guidelines for sample collection and serology testing when screening: (i) influenza vaccine immunogenicity antibody response; (ii) antibody responses to newly emerging viral strains; and (iii) clinical samples for anti-influenza antibody activity.

Abstract 4598: A comprehensive assessment of the impact of preanalytical variables on cell free DNA and circulating tumor cells in blood

Abstract Liquid biopsy continues to gain traction as a minimally invasive method to monitor biomarkers associated with malignancy and metastasis. A simple blood draw can reveal key biomarkers such as circulating tumor cells (CTCs) and cell-free DNA (cfDNA). However, these analytes are present in low quantities and are prone to rapid degradation and contamination, which presents challenges for the detection of rare CTCs and cfDNA targets. Higher molecular weight genomic DNA from poorly stabilized leukocytes can contaminate plasma, compromising liquid biopsy assay results by increasing the total amount of extracted DNA while diluting rare cfDNA targets. Preservation of CTCs and cfDNA over time is thus essential for the accuracy of downstream tests, and preanalytical variables that impact plasma volume, cfDNA yield and quality, and cell recovery in ex vivo blood must be understood. Here, we identified several preanalytical variables, including type of blood collection tube, storage time and conditions post-phlebotomy, and centrifugation conditions for plasma fractionation. We then carried out a series of studies to better understand the interplay between these preanalytical variables and plasma recovery, cell recovery, and cfDNA preservation in healthy donor blood, contrived samples, and patient samples. We found that choice of blood collection tube, in particular, has a significant impact on preservation of plasma volume, CTCs, cfDNA over time, particularly under real-world stress conditions including temperature excursions, sample shipping, and storage time post-draw. The work presented here demonstrates the importance of assessing preanalytical variables for the preservation of cancer biomarkers in whole blood without compromising sample integrity. This will ultimately help researchers make informed decisions on how best to stabilize their analytes of interest without introducing un-intended variables due to blood tube selection, shipping and storage conditions, and plasma isolation conditions. Citation Format: Anita Pottekat, Hatim T. Allawi, Genna T. Boragine, Michael W. Kaiser, Tamara Sander, Chateen Krueger, Janelle J. Bruinsma, Amber N. Murray. A comprehensive assessment of the impact of preanalytical variables on cell free DNA and circulating tumor cells in blood [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4598.

Surface characterization and free thyroid hormones response of chemically modified poly(ethylene terephthalate) blood collection tubes

The surface chemistry and surface energy of chemically modified polyethylene terephthalate (PET) blood collection tubes (BCTs) were studied and the results showed a significant increase in hydrophilicity and polarity of modified PET surface. The surface modification created nanometer-sized, needle-like asperities through molecular segregation at the surface. The surface dynamics of the modified PET was examined by tracking its surface properties over a 280-day period. The results showed surface rearrangement toward a surface with lower surface energy and fewer nanometer-sized asperities. Thromboelastography (TEG) was used to evaluate and compare the thrombogenicity of the inner walls of various types of BCTs. The TEG tracings and data from various types of BCTs demonstrated differences in the reactionand coagulation times but not in clot strength. The performance of the modified tubes in free triiodothyronine (FT3) and free thyroxine (FT4) hormone tests was examined, and it was found that the interference of modified PET tubes was negligible compared to that of commercially available PET BCTs.

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.

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.