Type Of Collection Tube Research Articles

The procurement, storage, and quality assurance of frozen blood and tissue biospecimens in pathology, biorepository, and biobank settings

Well preserved frozen biospecimens are ideal for evaluating the genome, transcriptome, and proteome. While papers reviewing individual aspects of frozen biospecimens are available, we present a current overview of experimental data regarding procurement, storage, and quality assurance that can inform the handling of frozen biospecimens. Frozen biospecimen degradation can be influenced by factors independent of the collection methodology including tissue type, premortem agonal changes, and warm ischemia time during surgery. Rapid stabilization of tissues by snap freezing immediately can mitigate artifactually altered gene expression and, less appreciated, protein phosphorylation profiles. Collection protocols may be adjusted for specific tissue types as cellular ischemia tolerance varies widely. If data is not available for a particular tissue type, a practical goal is snap freezing within 20min. Tolerance for freeze–thaw events is also tissue type dependent. Tissue storage at −80°C can preserve DNA and protein for years but RNA can show degradation at 5years. For −80°C freezers, aliquots frozen in RNAlater or similar RNA stabilizing solutions are a consideration. It remains unresolved as to whether storage at −150°C provides significant advantages relative to that at −80°C. Histologic quality assurance of tissue biospecimens is typically performed at the time of surgery but should also be conducted on the aliquot to be distributed because of tissue heterogeneity. Biobanking protocols for blood and its components are highly dependent on intended use and multiple collection tube types may be needed. Additional quality assurance testing should be dictated by the anticipated downstream applications.

Effect of Pre-Analytical Conditions on the Thrombodynamics Assay

Standardization of pre-analytical conditions is the obligatory step for all potential diagnostic tests. Spatial clot growth (Thrombodynamics) is a new global hemostasis assay that considers spatial organization of coagulation. The principal parameter is rate of fibrin clot growth from the tissue-factor coated surface. In this work we studied the pre-analytical variables of Thrombodynamics assay that include conditions of blood collection, sample preparation and storage. Blood of apparently healthy volunteers was used. Eight types of citrate blood collection tubes were tested, centrifugation conditions for plasma preparation were evaluated and impact of plasma freezing/thawing was tested. Among the blood collection tubes tested, BD Vacutainer glass tubes showed a significantly higher clot growth rate compared to plastic tubes. There was no difference between 3.2% and 3.8% of sodium citrate. For plasma preparation, a single 15 min centrifugation at 1,600 g shows significantly increased clot growth rate compared to plasma obtained by two sequential centrifugations (15 min 1 600 g, 5min 10,000 g). There was no significant difference between 1,600 g and 2,100 g if the second centrifugation was performed. For the second centrifugation there was no difference between 20 min at 1 600 g and 5 min at 10,000g. Frozen-thawed plasma showed increased clot growth rate compared to fresh plasma. The data represent the necessary steps for the standardization of Thrombodynamics assay and for the formulation of the operating guide.

Accurate use of neutrophil/lymphocyte ratio from the perspective of laboratory experts

Dear editor I read with great interest the recent published article “Neutrophil/lymphocyte ratio in patients with atrial septal aneurysm” by Demir and Demir.1 In this study, researchers compared the neutrophil/lymphocyte ratio (NLR) between patients with atrial septal aneurysm (ASA) and controls. It was reported that patients with ASA have higher NLR compared with controls.1 There are some points that we would like to address from this study. Firstly, a white blood cell (WBC) count of more than 12,000 cells per μL, or less than 4,000 cells per μL, was mentioned as one of the exclusion criteria.1 However, the authors did not state why these values were used as the limits for inclusion. Whether a WBC count of more than 12,000 cells per μL is accepted as an indicator of inflammation or infection is unclear, and there is no mention of the literature that this value is based on, or which device (brand/model) was used for the WBC count. The WBC reference ranges may vary depending on many factors such as the population studied, the individual laboratory, and the instruments (eg, types of collection tubes) or measurement methods used (eg, waiting period prior to analysis).2,3 Secondly, despite comparing groups based on neutrophil and lymphocyte counts, there was no exclusion criteria specified for these parameters. According to Horne et al,4 and as stated in the study,1 high neutrophil, monocyte, and NLR counts, as well as low lymphocyte counts, are independently related to increased cardiovascular events. However, in this study, Table 2 shows an increased total lymphocyte count (TLC) in controls (2,911±837 cells per mm3) draws attention rather than the low TLC of patients (1,862±625 cells per mm3).1 A TLC significantly higher than 2,900 lymphocytes in a μL of blood is generally considered to be an indicator of lymphocytosis in adults.3 If you accept 2,900 cells per mm3 as the upper limit of the TLC, the differential diagnosis of lymphocytosis should be given to the controls included in the study. However, in several other sources 4,000 cells per mm3 is regarded as the upper limit of the TLC.5,6 Additionally, although the TLC obtained from the patient group was relatively lower than in the control group, all results were within the reference values.3 Therefore, it would not be correct to declare that the patient group had a low TLC. On the basis of this information, evaluation of the percentage of lymphocytes and neutrophils may also be important in patient groups with borderline TLC values. Automated blood counting instruments should provide percentage values for each type of WBC count. Thirdly, the measurement of NLR was suggested as an indicator of increased risk of arrhythmia, based on the association found between ASA and NLR in the study, however the physiopathological causes supporting this conclusion were not thoroughly discussed.1 NLR has been used frequently to predict outcomes in patients with cancer and coronary artery disease.7,8 Systemic inflammation is thought to be a risk factor for cardiovascular disease, and NLR, which integrates the detrimental effects of neutrophilia (an indicator of inflammation) and lymphopenia (an indicator of physiological stress), has emerged as a useful prognostic marker.8 Possible causes affecting neutrophil/lymphocyte counts and the confounding factors which have a considerable effect on the clinical availability of NLR should be discussed in more detail in this study population. Finally, NLR itself alone without other inflammatory markers may not accurately provide information about the prognosis of the patients. There are studies evaluating soluble P- and E-selectin, interleukin-6, and high-sensitivity C-reactive protein (hs-CRP) as indices of prothrombogenic and proinflammatory activity in a similar group of patients.9,10 While assessing the predictive values of parameters like NLR (derived from the ratios), and comparing them for validity and accuracy, proven markers will provide more reliable results every time. In conclusion, the types of collection tubes, waiting period prior to analysis, instrumental parameters and reference ranges for each parameter must be specified as they are easily affected by analytical and preanalytical variables in studies based on laboratory results.11

Blood Collection Tubes Influence Serum Ficolin-1 and Ficolin-2 Levels

The ficolins are members of a recently discovered family of host innate opsonins that can activate the lectin pathway of complement. The ficolins bind many ligands, although they are typically described as binding acetylated sugars. Ficolin-1 (M-ficolin) and ficolin-2 (L-ficolin) are known to bind Streptococcus pneumoniae serotypes 19C and 11A, respectively. While studying the binding of ficolins to pneumococci, we found variations in ficolin-2 binding among serum samples collected in different types of blood collection tubes. Plastic tubes, which contain a silica clot activator, yielded sera with reduced ficolin-2 binding and apparent ficolin-2 levels. We found that the silica clot activator eluted from plastic red-top tubes inhibited ficolin-2 ligand binding, while other related proteins, like mannose-binding lectin (MBL) and ficolin-1, were not affected. These tube types did not affect the concentrations of other related opsonins (C1q, MBL, or ficolin-3 [H-ficolin]). Interestingly, we also found that ficolin-1 levels were increased 2- to 3-fold in plastic serum separator tubes compared to the increases in other tube types. These findings have implications for future ficolin-1 and ficolin-2 studies, as proper sample collection and handling are essential.

Impact of harmonization of collection tubes on Alzheimer's disease diagnosis

ObjectiveThe objective of this study was to analyze differences in biomarker outcomes before and after harmonization of cerebrospinal fluid (CSF) collection tubes in Alzheimer's disease (AD) diagnosis. MethodsWe analyzed data from French memory centers that switched from different CSF collection tubes to a common one. A total of 1966 patients were included in the study. CSF concentrations of β-amyloid 1–42 (Aβ42), total tau, and phosphorylated tau (p-tau181) were measured in each center using the same commercial enzyme-linked immunoabsorbent assay (ELISA) kits. The diagnostic value of CSF biomarkers according to the type of tube used was then assessed using different cutoffs. ResultsThe predictive value of Aβ42 was highly affected by the type of collection tube used. The optimal cutoff value for p-tau181 appeared not to be affected by the type of collection tube whereas that of total tau was slightly changed. New optimal cutoff values were then computed. ConclusionsIn a routine clinical environment, the selection of the collection tube and biomarker cutoff value makes a major difference in AD biological diagnosis. The use of a common collection tube among different centers will reduce the risk of misdiagnosis and incorrect patient stratification.

Effect of blood handling conditions on progesterone assay results obtained by chemiluminescence in the bitch

Assay of blood progesterone (P4) is commonly practiced to determine the time of ovulation, diagnose luteal insufficiency, and predict time of parturition in bitches. Because of practical constraints, most blood samples cannot be assayed on site immediately after collection. The aim of this work was to evaluate the effects of various sampling and storage conditions on concentrations of P4 as determined by chemiluminescence immunoassay. The blood of 5 Beagle bitches was collected from the jugular vein to study the effect of the type of collection tube (silicone, lithium heparin, EDTA), the storage time of unseparated or separated plasma (2 h to 14 d), and the number of freeze-thaw cycles (1–10) on P4. The effect of each factor was tested within one assay session. None of the factors significantly affected P4. Thus, P4 appears to remain relatively stable in canine blood samples exposed to various processing and storage conditions.

Serum fibrinogen alpha C‐chain 5.9 kDa fragment as a biomarker for early detection of hepatic fibrosis related to hepatitis C virus

Clinical application of biomarker candidates discovered by proteomic analysis is challenging. The purpose of this study was to standardize preanalytical conditions for measurement of serum levels of fibrinogen alpha C-chain 5.9 kDa fragment (FIC 5.9) and to test the diagnostic value of this peptide for detection of early hepatic fibrosis in patients with hepatitis C virus (HCV)-related chronic hepatitis. Serum FIC 5.9 levels were measured by a sandwich ELISA. Effects on the serum FIC 5.9 level of temperature, the time between venipuncture and serum separation, and the types of collection tubes used were examined. The diagnostic value of serum FIC 5.9 as an early indicator of hepatic fibrosis due to HCV was then assessed. FIC 5.9 was produced in a time- and temperature-dependent manner after venipuncture. Abnormal FIC 5.9 values were found in 89.5% of FI stage patients. Receiver operating characteristic analyses confirmed the superiority of FIC 5.9 over other conventional markers for early detection of fibrosis. The serum FIC 5.9 level may be an early indicator of hepatic fibrosis in HCV-related chronic liver diseases. This study provides an example of a pipeline from biomarker discovery by proteome analysis to assay optimization and preliminary clinical validation.

Preanalytic variables of thrombin generation: towards a standard procedure and validation of the method

Thrombin generation assays are sensitive methods for assessment of the overall clotting potential of plasma, but, despite their common use in thrombosis research, standardization of preanalytic conditions is lacking. In order to set up a standardized protocol, we analyzed different preanalytic variables and validated the calibrated automated thrombogram method. Thrombin generation was assessed with 0, 1 and 5 pm tissue factor (TF). Variations in thrombin generation were mostly attributable to the type of collection tube, mainly because of variations in contact activation. The collection tube also determined the influence of other preanalytic variables on thrombin generation, e.g. the need for a discard tube, the storage of whole blood, and the centrifugation method. Regarding the collection system, blood drawn through intravenous catheters or butterfly needles showed significantly more hemolysis than blood obtained with venipuncture using conventional needles. The results showed that a discard tube is still needed for thrombin generation measurements. After blood collection, whole blood is best centrifuged immediately, to prevent activation or degradation of coagulation proteins, and a second centrifugation step at 10,000 × g is recommended. After thawing, plasma is best analyzed immediately, as storage resulted in thrombin generation results outside the 10% range of the reference sample. On the basis of these results, we set up an in-house standardized protocol, which was used for validation, resulting in coefficients of variations of < 15% for all derived parameters with both the 1 and 5 pm TF triggers. Thrombin generation was greatly influenced by preanalytic conditions, demonstrating the need for an international standardized protocol.

Sensitive PCR-based quantitation of cell-free circulating microRNAs

Cell-free microRNAs (miRNAs) that circulate in the blood are promising surrogate biomarkers of disease and physiological processes. The ease of quantifying specific miRNA species using made-to-order approaches based on Taq-polymerase has led to numerous studies that have identified changes in the abundance of circulating cell-free miRNA species that correlate with pathology or other events. The growing interest in developing miRNAs as blood biomarkers necessitates the careful consideration of the unique properties of such body fluids that can make the reproducible and quantitative assessment of RNA abundance challenging. For example, enzymes involved in the amplification and analysis of RNA can be affected by blood components that copurify with miRNA. Thus, if miRNAs are to be effectively utilized as biomarkers, it is important to establish standardized protocols for blood collection and miRNA analysis to ensure accurate quantitation. Here we outline several considerations, including the type of collection tube used in sampling, the influence of added anticoagulants and stabilizers, sample processing, enrichment of vesicular and other miRNA species, RNA extraction approaches and enzyme selection, that affect quantitation of miRNA isolated from plasma and should be considered in order to achieve reproducible, sensitive and accurate quantitation.

The Effect of Pre-Analytical Variability on the Measurement of MRM-MS-Based Mid- to High-Abundance Plasma Protein Biomarkers and a Panel of Cytokines

Blood sample processing and handling can have a significant impact on the stability and levels of proteins measured in biomarker studies. Such pre-analytical variability needs to be well understood in the context of the different proteomics platforms available for biomarker discovery and validation. In the present study we evaluated different types of blood collection tubes including the BD P100 tube containing protease inhibitors as well as CTAD tubes, which prevent platelet activation. We studied the effect of different processing protocols as well as delays in tube processing on the levels of 55 mid and high abundance plasma proteins using novel multiple-reaction monitoring-mass spectrometry (MRM-MS) assays as well as 27 low abundance cytokines using a commercially available multiplexed bead-based immunoassay. The use of P100 tubes containing protease inhibitors only conferred proteolytic protection for 4 cytokines and only one MRM-MS-measured peptide. Mid and high abundance proteins measured by MRM are highly stable in plasma left unprocessed for up to six hours although platelet activation can also impact the levels of these proteins. The levels of cytokines were elevated when tubes were centrifuged at cold temperature, while low levels were detected when samples were collected in CTAD tubes. Delays in centrifugation also had an impact on the levels of cytokines measured depending on the type of collection tube used. Our findings can help in the development of guidelines for blood collection and processing for proteomic biomarker studies.

Impact of pre‐analytical parameters on the measurement of circulating microparticles: towards standardization of protocol

Microparticles (MP) are small vesicles of 0.1-1 μm, released in response to activation or apoptosis. Over the past decade, they received an increasing interest both as biomarkers and biovectors in coagulation, inflammation and cancer. Clinical studies were conducted to assess their contribution to the identification of patients at cardiovascular risk. However, among the limitation of such studies, pre-analytical steps remains an important source of variability and artifacts in MP analysis. Because data from the literature are insufficient to establish recommendations, the objective of the present study was to assess the impact of various pre-analytical parameters on MP measurement. These parameters included the type of collection tube, phlebotomy conditions, transportation practices, centrifugation steps and freezing. MP were assessed by three methods: flow cytometry using a standardized approach, a thrombin generation test (Calibrated Automated Thrombogram(®)) and a procoagulant phospholipid-dependent clotting time assay (STA(®) -Procoag-PPL). The main results show that the three major pre-analytical parameters which impact on MP-related data are the delay before the first centrifugation, agitation of the tubes during transportation and the centrifugation protocol. Based on both this work and literature data, we propose a new protocol that needs to be validated on a larger scale before being applied for multicenter studies.

Eosinophil Cationic Protein in Children With Respiratory Allergies - When Is It Useful?

Background: To assess the differences in serum eosinophilic cationic protein (ECP) concentrations between treated and untreated children with asthma, children with rhinitis, and children with both and possible influence of seasonal exposure to sensitizing allergens on ECP levels. Methods: The study included treated (n=156) and untreated (n=55) children with asthma, children with rhinitis, and children with asthma and rhinitis. Serum ECP was measured in serum collected between 8 am and 12 pm under standardized pre-analytical conditions (regarding the type of blood collection tube, time, and incubation temperature during blood clotting). Results: Untreated children had significantly higher ( P <0.0001) concentrations of ECP (M[IQR]=35.1 [29.5–50.9] μg/L) than treated children (M[IQR]=11.3 [7.1–16.1] μg/L). Eosinophilic cationic protein was significantly higher during the allergen exposure season (M[IQR]=23.9 [17.6–40] μg/L) than out of season (M[IQR]=8.3 [5.4–17.2]) μg/L, P =0.0001. Conclusions: To make ECP measurements useful in clinical practices, it is necessary to meet standardized pre-analytical conditions. * ECP : eosinophilic cationic protein IL-2 : interleukin-2 ARIA : Allergic Rhinitis and its Impact on Asthma GINA : Global Initiative for Asthma IgE : immunoglobulin E FeNO : fraction of exhaled nitric oxide

Performance Characteristics of Six Intact Parathyroid Hormone Assays

The aim of this study was to evaluate the performance characteristics of 6 intact parathyroid hormone assays: Access 2 (Beckman Coulter, Fullerton, CA), ARCHITECT i2000(SR) (Abbott Diagnostics, Abbott Park, IL), ADVIA Centaur (Siemens Healthcare Diagnostics, Deerfield, IL), Modular E170 (Roche Diagnostics, Indianapolis, IN), IMMULITE 2000 (Siemens Healthcare Diagnostics), and LIAISON (DiaSorin, Stillwater, MN). Sample collection tubes and storage conditions were compared. Imprecision studies were performed using commercial quality control materials. Linearity was assessed using pools prepared from samples. For method comparison, serum and EDTA plasma samples were tested by all methods, and the ARCHITECT was used as the comparison method. Reference intervals were determined using various vitamin D cutoffs. The types of collection tubes and storage conditions are more important for some methods than others. Total coefficients of variation were 10.9% or less. The maximum deviation from the target recovery for linearity ranged from 5.0% to 82.2%. Bland-Altman plots demonstrated percentage biases ranging from -36.3% to 24.4%. The lower limit of the reference interval was not influenced by vitamin D status, whereas the upper reference limit was affected.

Effect of storage temperature or collection tube type on blood methylmalonic acid concentrations measured by gas chromatography-mass spectrometry

Effect of storage temperature or collection tube type on blood methylmalonic acid concentrations measured by gas chromatography-mass spectrometry

Evaluation of blood collection tubes using selected reaction monitoring MS: Implications for proteomic biomarker studies

An emphasis of current proteomic research is the validation of plasma protein biomarkers. The process of blood collection itself is critical to the accuracy and reproducibility of quantitative biomarker assays. We have developed selected reaction monitoring (SRM) assays to analyse thirteen abundant plasma proteins and evaluated the impact of three different blood collection tubes on the levels of these proteins. We also assessed the implications of the time taken to analyse plasma samples by evaluating the recovery of these proteins. We showed that SRM detects minor differences in the levels of some proteins which can be attributed to collection tube type. The average recovery for 12 of 18 assays was higher for proteins that were collected in tubes containing protease inhibitors compared to conventional collection tubes. For five of the assays, the differential recovery was statistically significant. Delaying MS analysis of a freeze-thawed sample for 1 hour showed greatly reduced recovery of these analytes; however differences attributed to tube type were only evident at the baseline timepoint. Finally, we assessed the natural variation of circulating levels of these proteins in a cohort of seven healthy individuals. This study provides useful information for researchers contemplating blood collection for undertaking protein biomarker studies.

Stability of Ribavirin Concentrations Depending on the Type of Blood Collection Tube and Preanalytical Conditions

Ribavirin pharmacokinetic and exposure effect trials based on either plasma or serum concentrations have yielded diverging results. This study aimed to compare ribavirin concentrations in serum and plasma and to investigate the influence of blood collection and preanalytical conditions on ribavirin concentration stability. Blood samples from patients with hepatitis virus C and receiving ribavirin were collected in plain (dry) tubes, in tubes containing ethylenediaminetetra-acetic acid or lithium-heparinate, in Type II Serum Separating Tubes with clot activator, or Type II lithium heparinate Plasma Separating Tubes. Different time and temperature conditions were tested before and after blood centrifugation. Ribavirin was determined using liquid chromatography-dual mass spectroscopy. Multiple-way analysis of variance was used for statistical analyses. Ribavirin concentrations showed a higher interlaboratory variability in serum than in plasma. Results were fairly stable over 2 hours in whole blood collected in dry or ethylenediaminetetra-acetic acid tubes and very stable up to 24 hours in serum or plasma kept in gel-containing tubes after immediate centrifugation. When Plasma Separating II gel tubes were kept at +4 degrees C or at ambient temperature for up to 24 hours before centrifugation, ribavirin concentrations decreased by 1% to 8% and 12% to 18%, respectively. These results suggest that blood samples should be collected in gel-containing tubes and centrifuged immediately, after which the tubes can be kept at ambient temperature for the next 24 hours. In case of clinical constraints, Plasma Separating II gel tubes can be kept at +4 degrees C for a maximum of 2 hours before centrifugation with limited impact on the measured concentrations.

Comparison of complete blood counts in samples obtained from healthy dogs and cats by use of standard and microsample blood collection tubes

To compare results of a CBC performed on blood samples obtained from healthy dogs and cats by use of standard and microsample collection tubes. Evaluation study. 29 healthy client-owned animals (14 dogs and 15 cats). A blood sample (3 mL) was collected from each animal; 2.5 mL was transferred into a vacuum tube that contained sodium EDTA, and 0.5 mL was transferred into a microsample tube that contained sodium EDTA. Variables evaluated were total numbers of RBCs and WBCs, hemoglobin concentration, Hct, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration (MCHC), mean platelet volume, and plasma total protein concentration as well as neutrophil, lymphocyte, monocyte, eosinophil, basophil, and platelet counts. Results for the 2 types of tube in each species were compared by use of Pearson correlation coefficients, Passing-Bablok regression analysis, and Bland-Altman analysis. The Pearson correlation coefficient was low for basophil count in cats and moderate, high, or very high for all other variables. Constant and proportional biases were identified for MCHC in dogs by use of Passing-Bablok regression analysis, although the mean difference between types of blood collection tubes was small. No evidence of constant or proportional bias for any other variable was revealed by regression analysis or Bland-Altman analysis. Samples obtained from healthy dogs and cats by use of microsample blood collection tubes provided clinically equivalent CBC results, compared with results for samples obtained by use of standard blood collection tubes, and minimized the total sample volume collected for diagnostic testing.

Comparison of biochemical variables in plasma samples obtained from healthy dogs and cats by use of standard and microsample blood collection tubes

To compare results of biochemical analyses performed on plasma samples obtained from healthy dogs and cats by use of standard and microsample blood collection tubes. Evaluation study. 29 healthy client-owned animals (14 dogs and 15 cats). A blood sample (3 mL) was collected from each animal; 2.5 mL was transferred into a vacuum tube that contained lithium heparin, and 0.5 mL was transferred into a microsample tube that contained lithium heparin. Variables evaluated were albumin, bicarbonate, BUN, calcium, chloride, cholesterol, creatinine, glucose, phosphorus, potassium, sodium, total bilirubin, and total protein concentrations and alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, and creatine kinase activities. Results for the 2 types of tubes in each species were compared by use of Pearson correlation coefficients, Passing-Bablok regression analysis, and Bland-Altman analysis. Data were normally distributed, except for creatine kinase activity of cats. The Pearson correlation coefficient was minimal for total bilirubin concentration in cats and moderate, high, or very high for all other variables. Constant bias for cholesterol and glucose concentration in dogs was identified during Bland-Altman analysis, although the mean difference between types of blood collection tubes was small. No constant or proportional bias for any other variable was revealed by regression analysis or Bland-Altman analysis. Samples obtained from healthy dogs and cats by use of microsample blood collection tubes that contained lithium heparin provided clinically equivalent biochemical results, compared with results for samples obtained by use of standard blood collection tubes, and minimized the total sample volume collected for diagnostic testing.

The impact of delayed blood centrifuging, choice of collection tube, and type of assay on 25-hydroxyvitamin D concentrations

Studies have examined the associations between cancers and circulating 25-hydroxyvitamin D [25(OH)D], but little is known about the impact of different laboratory practices on 25(OH)D concentrations. We examined the potential impact of delayed blood centrifuging, choice of collection tube, and type of assay on 25(OH)D concentrations. Blood samples from 20 healthy volunteers underwent alternative laboratory procedures: four centrifuging times (2, 24, 72, and 96 h after blood draw); three types of collection tubes (red top serum tube, two different plasma anticoagulant tubes containing heparin or EDTA); and two types of assays (DiaSorin radioimmunoassay [RIA] and chemiluminescence immunoassay [CLIA/LIAISON((R))]). Log-transformed 25(OH)D concentrations were analyzed using the generalized estimating equations (GEE) linear regression models. We found no difference in 25(OH)D concentrations by centrifuging times or type of assay. There was some indication of a difference in 25(OH)D concentrations by tube type in CLIA/LIAISON((R))-assayed samples, with concentrations in heparinized plasma (geometric mean, 16.1 ng ml(-1)) higher than those in serum (geometric mean, 15.3 ng ml(-1)) (p = 0.01), but the difference was significant only after substantial centrifuging delays (96 h). Our study suggests no necessity for requiring immediate processing of blood samples after collection or for the choice of a tube type or assay.

The stability of the circulating human proteome to variations in sample collection and handling procedures measured with an aptamer-based proteomics array

Blood-based protein biomarkers hold great promise to advance medicine with applications that detect and diagnose diseases and aid in their treatment. We are developing such applications with our proteomics technology that combines high-content with low limits of detection. Biomarker discovery relies heavily on archived blood sample collections. Blood is dynamic and changes with different sampling procedures potentially confounding biomarker studies. In order to better understand the effects of sampling procedures on the circulating proteome, we studied three sample collection variables commonly encountered in archived sample sets. These variables included (1) three different sample tube types, PPT plasma, SST serum, and Red Top serum, (2) the time from venipuncture to centrifugation, and (3) the time from centrifugation to freezing. We profiled 498 proteins for each of 240 samples and compared the results by ANOVA. The results found no significant variation in the measurements for most proteins (approximately 99%) when the two sample processing times tested were 2h or less, regardless of sample tube type. Even at the longest timepoints, 20 h, approximately 82% of the proteins, on average for the three collection tube types, showed no significant change. These results are encouraging for proteomic biomarker discovery.